Bioluminescent plants and methods of making same

ABSTRACT

A genetically modified plant cell containing a heterologous nucleotide sequence encoding a bioluminescent polypeptide, which is expressed in amount sufficient to produce at least about 750,000 photons of visible light/mm 2 /second, is provided, as is a visibly bioluminescent transgenic plant, which contains such a genetically modified plant cell. Also provided is a recombinant nucleic acid molecule, which contains a plant translational enhancer operatively linked to a nucleotide sequence encoding a bioluminescent polypeptide. In addition, methods of producing a genetically modified plant cell that is visibly bioluminescent introducing a transgene encoding a bioluminescent polypeptide into a plant cell, whereby the bioluminescent polypeptide is expressed at a level that produces at least about 750,000 photons of visible light/mm 2 /second are provided, as are kits containing such visibly bioluminescent compositions.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates generally to plant biology and morespecifically to plants that are genetically modified to exhibitbioluminescence, and to compositions and methods for making suchgenetically modified bioluminescent plants.

BACKGROUND INFORMATION

Plants and plant products provide the primary sustenance, eitherdirectly or indirectly, for all animal life, including humans. For themajority of the world's human population and for many animals, plantsand plant products provide the sole source of nutrition. As the worldpopulation increases, the best hope to prevent widespread famine is toincrease the quantity and improve the quality of food crops.

Throughout history, a continual effort has been made to increase theyield and nutritious value of food crops. For centuries, plants havingdesirable characteristics such as greater resistance-to droughtconditions or increased size of fruit were crossbred and progeny plantsexhibiting the desired characteristics were selected and used to produceseed or cuttings for propagation. Using such classical genetic methods,plants having, for example, greater disease resistance, increased yield,and better flavor have been obtained.

More recently, the methods of genetic engineering have been applied toplant biology, thus expediting the generation of plants having desiredcharacteristics. For example, genetically engineered tomato plants havebeen generated such that the tomatoes do not continue to ripen afterthey have been picked from the plant. As a result, there is no need topick unripe tomatoes and hope that they attain an optimum level ofripeness at the time the consumer is ready to eat them. Instead, thetomatoes can be picked at a time when they are near their optimalripeness, then shipped to consumers for consumption. Geneticallyengineered plants that are less susceptible to freezing or to damage bybiological pests also have been generated. The availability of suchplants has been a boon to the agricultural industry, and provides ameasure of certainty that sufficient quantities of food will remainavailable in the future.

Methods of genetic engineering also have impacted the ornamental plantindustry. In addition to protecting ornamental plants from environmentalstresses or biological pests, genetic engineering has been used toproduce ornamental plants having new and interesting characteristics.For example, genetic engineering has been used to generate roses thatare blue in color. The ability to generate ornamental plants having newand unusual characteristics provides a means to expand the ornamentalplant market and, therefore, the economy in general. Thus, a need existsto identify interesting and unusual traits that can be expressed by aplant and can create a new market in the ornamental plant industry. Thepresent invention satisfies this need and provides additionaladvantages.

SUMMARY OF THE INVENTION

The present invention relates to a genetically modified plant cell thatexhibits bioluminescence that is visible to the naked eye. Such agenetically modified plant cell contains a heterologous nucleotidesequence that encodes a bioluminescent polypeptide, which, uponexpression, can result in the production of at least about 750,000photons of visible light/mm²/second by the plant cell. Thebioluminescent polypeptide can be any polypeptide that isbioluminescent, or can act in concert with a second molecule such thatvisible light is produced. In one embodiment, the genetically modifiedplant cell contains a heterologous nucleotide sequence encodingluciferase or a luciferase variant, for example, luc⁺. Upon contact of agenetically modified plant expressing luciferase or a variant thereofwith luciferin, a sufficient amount of visible light is produced suchthat the light is visible to the naked eye. Such a genetically modifiedplant cell generally expresses at least about 100 pg luc⁺/μg protein asdetermined by western blot analysis such that, upon contact of the plantcell with luciferin, the cell is visibly bioluminescent.

In another embodiment, the genetically modified plant cell, or the plantcell from which the genetically modified plant cell is derived, iscontacted with an agent that reduces or inhibits pigment formation inthe plant, for example, carotenoid synthesis, thereby reducing orinhibiting chlorophyll synthesis. Since chlorophyll in a plant cell canabsorb photons generated by the luciferase in the genetically modifiedplant cell, contact of the genetically modified plant cell, or atransgenic plant derived therefrom, with an amount of a carotenoidinhibitor sufficient to reduce or inhibit chlorophyll synthesis providesa means to enhance photon emission from the plant cell and, therefore,enhance the visible bioluminescence.

In still another embodiment, a visibly bioluminescent transgenic plantis generated from the genetically modified plant cell containing aheterologous nucleotide sequence encoding a bioluminescent polypeptide.As such, the invention relates to such a transgenic plant, as well as toa plant cell or tissue obtained from the transgenic plant, a seedproduced by the transgenic plant, and a cDNA or genomic DNA libraryprepared from the transgenic plant or from a plant cell or plant tissueobtained from the transgenic plant. The transgenic plant can be amonocot, or can be a dicot, for example, an angiosperm such as a cerealplant, a leguminous plant, an oilseed plant, a hardwood tree, or anornamental plant such as a petunia, an orchid, a carnation or the like.

The present invention also relates to a recombinant nucleic acidmolecule, which comprises a translational enhancer, which enhancestranslation in a plant cell, operatively linked to a nucleotide sequenceencoding a bioluminescent polypeptide. The translational enhancer can beany translational enhancer, for example, a plant potyvirus translationalenhancer such as tobacco etch virus (TEV) translational enhancer, atobacco mosaic virus omega translational enhancer, or a translationalenhancer comprising a Kozak sequence. The encoded bioluminescentpolypeptide can be a luciferase polypeptide or variant thereof, forexample, the luc⁺ luciferase variant.

A recombinant nucleic acid molecule of the invention also can contain atranscriptional regulatory element, i.e., promoter, enhancer, etc., thatenhances transcription in a plant cell, for example, a plant virusenhancer such as a cauliflower mosaic virus (CaMV) 35S enhancer, or aregulatory element from any other organism, for example, an actin 2regulatory element or a metallothionein regulatory element, whichcomprises a promoter and, optionally, an enhancer, wherein thetranscriptional regulatory element is operatively linked to the planttranslational enhancer and nucleotide sequence encoding thebioluminescent polypeptide. In one embodiment, a recombinant nucleicacid molecule contains, in operative linkage, a CaMV 35S enhancer, aCaMV 35S promoter, a TEV translational enhancer, a nucleotide sequenceencoding a luciferase polypeptide or variant thereof, and a CaMV 35Sterminator. In another embodiment, the CaMV 35S enhancer is a dual CaMV35S enhancer, and the luciferase polypeptide or variant thereof is theluc⁺ luciferase variant. Such a recombinant nucleic acid molecule isexemplified herein by the nucleotide sequence set forth as nucleotides8366 to 11,113 of SEQ ID NO:2. In still other embodiments, thetranscriptional regulatory element in a recombinant nucleic molecule ofthe invention is a metallothionein regulatory element or an actin 2regulatory element, and the translational enhancer is an omega enhancer,for example, in a recombinant nucleic acid molecule having a sequenceset forth as nucleotides 8340 to 12,098 of SEQ ID NO:4 or as nucleotides6 to 3570 of SEQ ID NO:5.

The present invention further relates to a vector containing arecombinant nucleic acid molecule comprising a translational enhanceractive in a plant operatively linked to a nucleotide sequence encoding abioluminescent polypeptide. Preferably, the vector is an expressionvector. In one embodiment, the recombinant nucleic acid moleculecontained in the vector comprises, in operative linkage, a CAMV 35Senhancer, a CaMV 35S promoter, a TEV translational enhancer, anucleotide sequence encoding a luciferase polypeptide or variantthereof, and a CaMV 35S terminator. In another embodiment, therecombinant nucleic acid molecule contained in the vector comprises, inoperative linkage, a dual CaMV 35S enhancer, a CaMV 35S promoter, a TEVtranslational enhancer, a nucleotide sequence encoding a luc⁺ luciferasevariant, and a CaMV 35S terminator. In still another embodiment, therecombinant nucleic acid molecule contained in the vector comprises thenucleotide sequence set forth as nucleotides 8366 to 11,113 of SEQ IDNO:2. In yet another embodiment, the transcriptional regulatory elementin the recombinant nucleic acid molecule comprises an actin 2 promoteror a metallothionein promoter, and the translation enhancer is an omegaenhancer, for example, in the recombinant nucleic acid molecules setforth as nucleotides 8340 to 12,098 of SEQ D) NO:4 or as nucleotides 6to 3570 of SEQ ID NO:5. Vectors of the invention are exemplified hereinby those as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 and SEQID NO:5. The present invention also provides a cell containing arecombinant nucleic acid molecule of the invention, or containing avector of the invention.

The present invention further relates to a method of producing agenetically modified plant cell that is visibly bioluminescent. A methodof the invention can be performed, for example, by introducing atransgene comprising a nucleotide sequence encoding a bioluminescentpolypeptide into a plant cell, whereby the bioluminescent polypeptide isexpressed at a level that produces at least about 750,000 photons ofvisible light/mm²/second. The transgene useful in a method of making agenetically modified plant cell that is visibly bioluminescent generallyincludes a translational enhancer that is active in a plant, forexample, a plant translational enhancer such as a plant potyvirustranslational enhancer (e.g., a TEV translational enhancer), a tobaccomosaic virus (TMV) translational enhancer such as the omega enhancer,operatively linked to the nucleotide sequence encoding a bioluminescentpolypeptide, for example, a luciferase polypeptide or variant thereof.The transgene also can contain a plant virus enhancer, for example, aCaMV 35S enhancer, which is operatively linked to the planttranslational enhancer and nucleotide sequence encoding a bioluminescentpolypeptide. In one embodiment, a transgene useful in making a visiblyluminescent genetically modified plant cell contains, in operativelinkage, a dual CaMV 35S enhancer, a CaMV 35S promoter, a TEVtranslational enhancer, a nucleotide sequence encoding a luciferasepolypeptide or variant thereof, and a CaMV 35S terminator. In anotherembodiment, the nucleotide sequence encodes a luc⁺ luciferase variantpolypeptide. Such transgenes are exemplified by the nucleotide sequencesset forth as nucleotides 8366 to 11,113 of SEQ ID NO:2, nucleotides 8340to 12,098 of SEQ ID NO:4, and nucleotides 6 to 3570 of SEQ ID NO:5.

A method of the invention can further include contacting the plant cell,or the genetically modified plant cell derived therefrom, with an agentthat inhibits the synthesis of a pigment such as carotenoids, which canabsorb light generated by the luciferase. For example, the geneticallymodified plant cell can be contacted with an agent such as norfluorazon,which reduces or inhibits chlorophyll production in the plants, thusallowing increased emission of photons from the plant cell, or from atrrnsgenic plant derived from the plant cell.

A visibly bioluminescent genetically modified plant cell made accordingto a method of the invention generally contains the transgene stablymaintained in the plant cell genome. Preferably, the transgene isintegrated into the plant cell genome. As such, the present inventionalso relates to a genetically modified plant cell produced by a methodof the invention, as well as to a transgenic plant containing orgenerated from such a genetically modified plant cell.

The present invention also relates to kit, which contains a geneticallymodified plant cell of the invention, or a derivative of the geneticallymodified plant cell, for example, a transgenic plant derived from thegenetically modified plant cell, or a cell, a tissue, or an organ ofsuch a transgenic plant. As such, a kit of the invention can contain oneor more flowers, bracts, leaves or other tissues or organs, which, uponcontact with luciferin, are visibly luminescent.

Accordingly, a kit of the invention can further include an amount ofluciferin sufficient for generating visible bioluminescence of thegenetically modified plant cell or the derivative of the geneticallymodified plant cell, and also can include a plurality of such amounts ofluciferin, thus allowing the generation of visible bioluminesce a numberof times, as desired. In addition, a kit of the invention can include anamount of a inhibitor that reduces or inhibits pigment synthesis in thegenetically modified plant cell or the derivative of the geneticallymodified plant cell. For example, the kit can contain a carotenoidsynthesis inhibitor such as norfluorazon, which reduces or inhibitschlorophyll production.

In another embodiment, a kit of the invention contains one or morecuttings, seeds, or other portion or derivative of a visiblybioluminescent plant of the invention such that a visibly luminescenttransgenic plant can be grown therefrom. As such, the kit also caninclude reagents for growing a transgenic plant from the cutting orseed, including, for example, a suitable plant food or other nutrientsource required for growth of the particular plant. In addition, the kitcan include an amount of a carotenoid inhibitor sufficient for reducingor inhibiting chlorophyll production in a trrnsgenic plant grown fromthe cutting or seed; and/or can include an amount of luciferinsufficient for generating visible bioluminescence of a transgenic plantgrown from the cutting or seed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a map of the pTK1829 plasmid. Restriction endonucleasesites are indicated, as are the first nucleotide positions of the sites,except that the first and last nucleotide positions of the multiplecloning site are indicated. “luc⁺” indicates region encoding variantluciferase polypeptide; “Amp” indicates ampicillin-resistance gene (see,also, SEQ ID NO:1).

FIG. 2 shows a map of the pPZPTK1829 binary vector plasmid. Restrictionendonuclease sites and nucleotide positions are indicated as in FIG. 1.“luc⁺” indicates region encoding variant luciferase polypeptide; “Spec”and “Gent-R” indicate the genes conferring resistance to spectinomycinand gentamycin, respectively. “Ori” indicates the plasmid origin ofreplication. “LB” and “RB” indicate left border and right border,respectively, of the binary vector that allow transfer of the insertinto the plant (see, also, SEQ ID NO:2).

FIG. 3 shows a map of the MT-OM-LUC⁺ binary vector plasmid. Restrictionendonuclease sites and nucleotide positions are indicated as in FIG. 1.“luc⁺”, “Spec”, “Gent-R”, “Ori”, and “LB” and “RB” are as in FIG. 2.“MT” indicates the metallothionein transcriptional regulatory element;“Ome” indicates the omega translational enhancer; and “E9 3′” indicatesthe RbcS E9 polyA region (see, also, SEQ ID NO:4).

FIG. 4 shows a map of the ACT-OM-LUC⁺ binary vector plasmid. Restrictionendonuclease sites and nucleotide positions are indicated as in FIG. 1.“luc⁺”, “Spec”, “Gent-R”, “ori”, “LB” and “RB” are as in FIG. 2. “Ome”and “E9 3′” are as in FIG. 3; “ACT” indicates the actin 2transcriptional regulatory element (see, also, SEQ ID NO:5).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides genetically modified plant cells that arevisibly bioluminescent due to expression of heterologous polypeptide,and visibly bioluminescent transgenic plants comprising or generatedfrom such genetically modified plant cells. As disclosed herein, thetransgenic plants of the invention exhibit bioluminescence that isvisible to the human eye. Prior to the present disclosure, plant cellscould be genetically modified to express heterologous bioluminescentpolypeptides such as luciferase, but light gathering or amplifyinginstrumentation was required to detect the bioluminescence; one couldnot simply look at plant comprising such cells directly and detect thebioluminescence. It is noted that there is a report on the world wideweb, at the URL “hybridorchids.com”, of a bioluminescent orchid.However, no published information for making such orchids is availableand the information on the website has not been updated since 2000. Thepresent invention provides compositions and methods that obviate theprevious limitations that prevented the generation of visibly detectableplants and plant cells. As disclosed herein, the visibly bioluminescentgenetically modified plant cells and transgenic plants of the inventionare useful as research tools, and have great ornamental value.

A genetically modified plant cell or transgenic plant of the inventionis visibly bioluminescent due to expression in the plant cell, or inplant cells of the transgenic plant, of a bioluminescent polypeptide. Asused herein, the term “bioluminescent polypeptide” refers to apolypeptide that can be expressed in a living organism and exhibits oreffects the emission of visible light. A bioluminescent polypeptide caninherently emit visible light such as fluorescence, or can be contactedwith a physical or chemical agent such that a reaction occurs andvisible light is produced. For example, a genetically modified plantcell can contain a heterologous nucleotide sequence encoding luciferasesuch that, upon expression of the luciferase and contact of the plantcell with luciferin, a chemiluminescent reaction occurs and visiblelight is emitted. As used herein, the term “visible light” refers toelectromagnetic radiation associated with the visible portion of theelectromagnetic spectrum, generally electromagnetic radiation having awavelength between about 100 to 1000 nanometers (nm), and particularlybetween about 350 to 750 nm.

Luciferase polypeptides and luciferase variants are examples ofbioluminescent polypeptides that are particularly useful for practicingthe methods and producing the compositions of the invention. As usedherein, the term “luciferase variant” refers to a modified luciferasepolypeptide that contains one or more amino acid additions, deletions orsubstitutions as compared to a corresponding wild type luciferasepolypeptide, and that, when contacted with an appropriate substrate suchas luciferin, undergoes a reaction that results in-the production ofvisible light.

Luciferases include bacterial luciferases such as Vibrio harveyi or V.fisheri luciferase, marine ostracod crustacean luciferases such aVargula hilgendorii luciferase, dinoflagellate luciferases such asGonyaulax ploydra luciferase, coelenterate luciferases such as Renillarenifornis (sea pansy) luciferase, or Coleoptera (beetle) luciferasessuch as an Elateridae (click beetle) family luciferase, a Phengodidae(glow worm) family luciferase, or a Lampyridae (firefly) luciferase, forexample, a Photintus carolinus or P. pyralis luciferase, and the like,including modified forms thereof (see, for example, U.S. Pat. No.4,968,613; U.S. Pat. No. 5,221,623; U.S. Pat. No. 5,229,285; U.S. Pat.No. 5,330,906; U.S. Pat. No. 5,604,123; U.S. Pat. No. 5,618,722; U.S.Pat. No 5,674,713; U.S. Pat. No. 5,814,465; and U.S. Pat. No. 5,843,746,each of which is incorporated herein by reference). A luciferase variantis exemplified by the firefly luciferase variant, luc⁺ (U.S. Pat. No.5,670,356; Sherf and Wood, Promega Notes 49:14, 1994, each of which isincorporated herein by reference), which lacks a peroxisome localizingdomain, can be particularly useful for preparing a genetically modifiedplant cell or visibly bioluminescent transgenic plant of the invention.Polynucleotide sequences encoding luciferases and mutant luciferases arewell known (see above references) and vectors containing suchpolynucleotides are commercially available (Promega; Madison Wis.).

As used herein, the term “visibly bioluminescent” refers to the emissionof a sufficient amount of visible light such that it can be seen by ahuman eye. As such, the use of light gathering, light amplifying, lightenhancing or light accumulating instrumentation is not required todetect the bioluminescence, although such instrumentation can be used,for example, to quantitate the amount of visible light emitted. Itshould be recognized that a visibly bioluminescent plant cell may not bevisible to the unaided eye due to its small size, but can be seen usinga magnifying glass, microscope, or the like. Such a plant cell isconsidered to be visibly bioluminescent for purposes of the presentinvention because light gathering, accumulating or amplifyinginstrumentation such as a luminometer, photomultiplier, image enhancingvideo system, spectrophotometer, film emulsion, digitizing equipment, orthe like is not required.

The light produced by a visibly bioluminescent transgenic plant can beseen by a person or other mammal looking at the plant, particularly whenthe plant is in a dark environment, for example, in a darkened room oroutside at night. In addition, a bioluminescent plant of the inventioncan be photographed using a camera, including any generally used camerasuch as a 35 mm single lens reflex camera, a digital camera, a Polaroid®camera, and the like, such that a picture of the glowing plant can beobtained. As disclosed herein, a genetically modified plant cell, or aplant, plant tissue or plant organ comprising the plant cell that emitsat least about 750,00 photons of visible light/mm²second, generally atleast about 850,000 photons of visible light/mm²/second, andparticularly at least about 1×10⁶ (one million) photons of visiblelight/mm²/second is visibly bioluminescent (see Example 2C, Table 4).

As disclosed herein, contact of a genetically modified plant cell of theinvention, or of a plant cell from which the genetically modified plantcell is derived, or of a transgenic plant of the invention with an agentthat reduces or inhibits the synthesis of one or more pigments in theplant or plant cell. By reducing the amount of pigmentation of a plant,or a portion of the plant such as the leaves or flowers, absorption ofbioluminescence by the pigment is reduced, thereby enhancing theemission of photons from the plant and, therefore, the visiblebioluminescence. For example, contact of the genetically modified plantcell or a transgenic plant of the invention with an inhibitor ofcarotenoid synthesis can reduce or inhibit chlorophyll synthesis in theplant, thus reducing absorption of photons by chlorophyll in the plantand allowing greater emission of the photons from the plant. Inhibitorsof carotenoid synthesis in plants are well known and include herbicidessuch as norfluorazone, which is commercially available as ZORIAL®herbicide (Syngenta Crop Production; Greensboro N.C.).

In another embodiment, a visibly bioluminescent transgenic plant isgenerated from the genetically modified plant cell containing aheterologous nucleotide sequence encoding a bioluminescent polypeptide.As such, the invention relates to such a transgenic plant, as well as toa plant cell or tissue obtained from the transgenic plant, a seedproduced by the transgenic plant, and a cDNA or genomic DNA libraryprepared from the trrnsgenic plant or from a plant cell or plant tissueobtained from the transgenic plant. The transgenic plant can be amonocot, or can be a dicot, for example, an angiosperm such as a cerealplant, a leguminous plant, an oilseed plant, a hardwood tree, or anornamental plant such as a petunia, a carnation or the like. In oneembodiment, plants useful for purposes of the present invention includeorchids. In another embodiment, plants useful for purposes of thepresent invention exclude orchids.

The present invention provides a recombinant nucleic acid molecule,which includes a heterologous nucleotide sequence encoding abioluminescent polypeptide operatively linked to one or moretranscriptional or translational regulatory elements, or a combinationthereof. As used herein, the term “heterologous” is used in acomparative sense with respect to a nucleotide sequence encoding abioluminescent polypeptide to indicate either that the nucleotidesequence is not an endogenous nucleotide sequence in a plant cell intowhich it is to be introduced, or that the nucleotide sequence is part ofa construct such that it is in a form other than it normally would befound in a plant cell in nature. For example, a heterologous nucleotidesequence can encode a firefly luciferase, which is not normallyexpressed in a plant cell and, therefore, is heterologous with respectto the plant cell.

The regulatory element or combination of regulatory elements is selectedsuch that the bioluminescent polypeptide can be expressed in a plantcell at a level sufficient for the plant cell, or a plant comprising thecell, to visibly bioluminesce. The term “regulatory element” is usedbroadly herein to refer to a nucleotide sequence that, when operativelylinked to a second, expressible nucleotide effects transcription ortranslation of the nucleotide sequence such that a ribonucleic acid(RNA) molecule or polypeptide, respectively, can be generated, orencodes a domain that confers a desirable characteristic on apolypeptide containing the domain. For purposes of the presentinvention, a regulatory element generally increases the amount oftranscription or translation of an operatively linked nucleotidesequence encoding the bioluminescent polypeptide, or provides a means tolocalize a bioluminescent polypeptide to a particular location in acell, particularly a plant cell. Transcriptional and translationalregulatory elements are well known and include promoters, enhancers,3′-untranslated or 5′-untranslated sequences of transcribed sequence,for example, a poly-A signal sequence or other transcription terminationsignal, or other protein or RNA stabilizing elements, or other geneexpression control elements known to regulate gene expression or theamount of expression of a gene product. A regulatory element useful forconstructing a recombinant nucleic acid molecule of the invention can beisolated from a naturally occurring genomic DNA sequence or can besynthetic, for example, a synthetic promoter.

Transcriptional regulatory elements can be constitutively expressedregulatory elements, which maintain gene expression at a relativelyconstant level of activity, or can be inducible regulatory elements.Constitutively expressed regulatory elements can be expressed in anycell type, for example an actin promoter such as an actin 2 promoter, oran elongation factor (EF) promoter such as an EF1α promoter; or can be atissue specific regulatory element, which is expressed only in one or afew specific cell types, a phase specific regulatory element, which isexpressed only during particular developmental or growth stages of aplant cell, or the like. A regulatory element such as a tissue specificor phase specific regulatory element or an inducible regulatory elementuseful in constructing a recombinant nucleic acid molecule of theinvention can be a regulatory element that generally, in nature, isfound in a plant genome. However, the regulatory element also can befrom an organism other than a plant, including, for example, from aplant virus, an animal virus, or a cell from an animal or othermulticellular organism.

As used herein, the term “tissue specific or phase specific regulatoryelement” means a nucleotide sequence that effects transcription in onlyone or a few cell types, or only during one or a few stages of the lifecycle of a plant, for example, only for a period of time during aparticular stage of growth, development or differentiation. The terms“tissue specific” and “phase specific” are used together herein inreferring to such a regulatory element because a single regulatoryelement can have characteristics of both types of regulatory elements.For example, a regulatory element active only during a particular stageof plant development also can be expressed only in one or a few types ofcells in the plant during the particular stage of development. A tissuespecific or phase specific regulatory element can be useful forproducing a genetically modified plant that is bioluminescent only inparticular organs or tissues, or only at a particular developmentalstage.

Numerous tissue specific or phase specific regulatory elements have beendescribed and are known and available to those in the art. Such tissueor phase specific regulatory elements include, for example, theAGL8/FRUITFULL regulatory element, which is activated upon floralinduction (Hempel et al., Development 124:3845-3853, 1997, which isincorporated herein by reference); root specific regulatory elementssuch as the regulatory elements from the RCP1 gene and the LRP1 gene(Tsugeki and Fedoroff, Proc. Natl. Acad. Sci., USA 96:12941-12946, 1999;Smith and Fedoroff, Plant Cell 7:735-745, 1995, each of which isincorporated herein by reference); flower specific regulatory elementssuch as the regulatory elements from the LEAFY gene and the APETELA1gene (Blazquez et al., Development 124:3835-3844, 1997, which isincorporated herein by reference; Hempel et al., supra, 1997); seedspecific regulatory elements such as the regulatory element from theoleosin gene (Plant et al., Plant Mol. Biol. 25:193-205, 1994, which isincorporated herein by reference), and dehiscence zone specificregulatory element. Additional tissue specific or phase specificregulatory elements include the Zn13 promoter, which is a pollenspecific promoter (Hamilton et al., Plant Mol. Biol. 18:211-218, 1992,which is incorporated herein by reference); the UNUSUAL FLORAL ORGANS(UFO) promoter, which is active in apical shoot meristem; the promoteractive in shoot meristems (Atanassova et al., Plant J. 2:291, 1992,which is incorporated herein by reference), the cdc2a promoter and cyc07promoter (see, for example, Ito et al., Plant Mol. Biol. 24:863, 1994;Martinez et al., Proc. Natl. Acad. Sci., USA 89:7360, 1992; Medford etal., Plant Cell 3:359, 1991; Terada et al., Plant J. 3:241, 1993;Wissenbach et al., Plant J. 4:411, 1993, each of which is incorporatedherein by reference); the promoter of the APETELA3 gene, which is activein floral meristems (Jack et al., Cell 76:703, 1994, which isincorporated herein by reference; Hempel et al., supra, 1997); apromoter of an agamous-like (AGL) family member, for example, AGL8,which is active in shoot meristem upon the transition to flowering(Hempel et al., supra, 1997); floral abscission zone promoters;L1-specific promoters; and the like. Such tissue specific or phasespecific regulatory elements can be used to construct a recombinantnucleic acid useful for producing, for example, a transgenic plant thatis visibly bioluminescent in one or a few selected tissues or organssuch as in flowers or in leaves, or that are expressed only during aparticular stage of development such as during flowering.

Inducible regulatory elements also are useful for constructing arecombinant nucleic acid molecule of the invention. As used herein, theterm “inducible regulatory element” means a regulatory element that,when exposed to an inducing agent, effects an increased level oftranscription of an operatively linked nucleotide sequence encoding thebioluminescent polypeptide as compared to the level of transcription, ifany, in the absence of an inducing agent. Inducible regulatory elementscan be those that have no basal or constitutive activity and only effecttranscription upon exposure to an inducing agent, or those that effect abasal or constitutive level of transcription, which is increased uponexposure to an inducing agent. Inducible regulatory elements that effecta basal or constitutive level of expression generally are particularlyuseful where the induced level of transcription is substantially greaterthan the basal or constitutive level of expression, for example, atleast about two-fold greater, or at least about five-fold greater.Particularly useful inducible regulatory elements do not have a basal orconstitutive activity, or increase the level of transcription at leastabout ten-fold greater than a basal or constitutive level oftranscription associated with the regulatory element.

The term “inducing agent” is used to refer to a chemical, biological orphysical agent that effects transcription from an inducible regulatoryelement. In response to exposure to an inducing agent, transcriptionfrom the inducible regulatory element generally is initiated de novo oris increased above a basal or constitutive level of expression. Suchinduction can be identified using the methods disclosed herein,including detecting an increased level of mRNA encoding thebioluminescent polypeptide. The use of an inducible regulatory elementin a recombinant nucleic acid molecule of the invention provides a meansto express the bioluminescent polypeptide only at a desired time, thuspreventing extraneous transcriptional or translational activity in theplant cell.

An inducing agent useful in a method of the invention is selected basedon the particular inducible regulatory element. For example, theinducible regulatory element can be a metallothionein (MT) regulatoryelement such as an MT2B regulatory element, a copper inducibleregulatory element, or a tetracycline inducible regulatory element, thetranscription from which can be effected in response to various metalions, to copper or to tetracycline, respectively (Furst et al., Cell55:705-717, 1988; Mett et al., Proc. Natl. Acad. Sci., USA 90:4567-4571,1993; Gatz et al., Plant J. 2:397-404, 1992; Roder et al., Mol. Gen.Genet. 243:32-38, 1994, each of which is incorporated herein byreference). The inducible regulatory element also can be an ecdysoneregulatory element or a glucocorticoid regulatory element, thetranscription from which can be effected in response to ecdysone orother steroid (Christopherson et al., Proc. Natl. Acad. Sci., USA89:6314-6318, 1992; Schena et al., Proc. Natl. Acad. Sci., USA88:10421-10425, 1991, each of which is incorporated herein byreference). In addition, the regulatory element can be a cold responsiveregulatory element or a heat shock regulatory element, the transcriptionof which can be effected in response to exposure to cold or heat,respectively (Takahashi et al., Plant Physiol. 99:383-390, 1992, whichis incorporated herein by reference). Additional regulatory elementsuseful in the methods or compositions of the invention include, forexample, the spinach nitrite reductase gene regulatory element (Back etal., Plant Mol. Biol. 17:9, 1991, which is incorporated herein byreference); a light inducible regulatory element (Feinbaum et al., Mol.Gen. Genet. 226:449, 1991; Lam and Chua, Science 248:471, 1990, each ofwhich is incorporated herein by reference), a plant hormone inducibleregulatory element (Yamaguchi-Shinozaki et al., Plant Mol. Biol. 15:905(1990); Kares et al., Plant Mol. Biol. 15:225 (1990), each of which isincorporated herein by reference), and the like. A circadian rhythmregulatory element such as a cab2 promoter (see, for example, Millar etal., Plant Cell 4:1075-1087, 1992, which is incorporated herein byreference) can also be considered an example of an inducible regulatoryelement, since expression from such a promoter appears to occur inresponse to a factor associated with a time of day.

A regulatory element operatively linked to a nucleotide sequenceencoding a bioluminescent polypeptide also can comprise an enhancer,including a transcriptional enhancer, translational enhancer, or both. Atranscriptional enhancer useful for purposes of the present inventioncan be any enhancer generally associated with a plant gene or anenhancer from another organism that has enhancer activity in a plantcell, for example, a plant virus enhancer, or an enhancer from a geneexpressed in an invertebrate cell, or a vertebrate cell such as amammalian cell, or an enhancer of a virus that infects such cells. Acauliflower mosaic virus (CaMV) 35S enhancer is an example of a plantvirus enhancer that is useful for increasing the transcriptionalactivity of a nucleotide sequence to which it is operatively linked.Thus, in one embodiment, a recombinant nucleic acid molecule of theinvention-contains a CaMV 35S enhancer operatively linked to anucleotide sequence encoding the bioluminescent polypeptide. Preferably,the CaMV 35S enhancer is a dual enhancer, comprising two copies of theCaMV 35S enhancer nucleotide sequence. An actin 2 regulatory elementprovides another example of a transcriptional regulatory element,comprising a promoter and an enhancer, that is useful in constructing acomposition of the invention or practicing a method of the invention(see nucleotides 6 to 1145 of SEQ ID NO:5). A metallothionein regulatoryelement provides another example of a transcriptional regulatory elementuseful for purposes of the present invention, and provides theadditional advantage that it includes an inducible enhancer (seenucleotides 11,000 to 12,098 of SEQ ID NO:4).

A translational enhancer, which enhances translation of a polypeptide ina plant cell (also referred to generally herein as a plant translationalenhancer), useful in a recombinant nucleic acid molecule of theinvention similarly can be any translational enhancer that generally isassociated with a plant gene or with a gene of another organism.Examples of a plant translational enhancer include a plant potyvirustranslational enhancer such as the tobacco etch virus (TEV)translational enhancer, and a tobacco mosaic virus translationalenhancer such as the omega enhancer (see, for example, nucleotides 1169to 1235 of SEQ ID NO:5; see, also, Carrington and Freed, J. Virol.64:1590, 1990; Gallie et al., Plant Cell 1:301-311, 1989, each of whichis incorporated herein by reference), as well as the nucleotide sequencebeginning about 101 nucleotide upstream of the potato virus S (PSV) coatprotein gene (Turner and Foster, Arch. Virol. 142:167-175, 1997, whichis incorporated herein by reference). A recombinant nucleic acidmolecule of the invention can also contain regulatory elements requiredfor termination of transcription, for example, a polyadenylation signal.A CaMV 35S terminator is an example of a transcription terminationsignal useful in constructing a recombinant nucleic acid molecule.

A recombinant nucleic acid molecule of the invention also can containregulatory elements required for initiation or termination oftranslation or both, including, for example, an internal ribosome entrysite (IRES), a Kozak sequence, a nucleotide sequence encoding aninitiator methionine residue, and a nucleotide sequence encoding a STOPcodon, which terminates translation. The regulatory element also can bea nucleotide sequence encoding a leader sequence, signal peptide, cellcompartmentalization domain, or the like, and can be included in theconstruct, for example, where it is desired to localize thebioluminescent polypeptide to a particular compartment such as thecytosol, nucleus, a chloroplast, or another subcellular organelle in aplant cell. Such intracellular localization can be particularly usefulwhere the genetically modified cell, or plant containing the cell, is tobe used as a research tool, for example, a particular intracellularcompartment of a plant cell.

A translation initiation or termination element or other regulatoryelement can be contained in the nucleotide sequence encoding thebioluminescent polypeptide can be a regulatory element that is normallyassociated with the nucleotide sequence in nature and is a part of thenucleotide sequence as a consequence of its isolation from a naturalsource, or can be heterologous to the nucleotide sequence andoperatively linked thereto. Additional regulatory elements such as anintron sequence, for example, from Adh1 or bronzel, or a viral leadersequence, for example, from TMV, MCMV or AIVIV, also enhance expressionof a nucleotide sequence encoding a bioluminescent polypeptide and,therefore, can be a component of a recombinant nucleic acid molecule ofthe invention.

As disclosed herein, combinations of regulatory elements can be selectedsuch that a recombinant nucleic acid molecule comprising a nucleotidesequence encoding a bioluminescent polypeptide can be constructed, andwherein the recombinant nucleic acid molecule allows for expression ofthe bioluminescent polypeptide in a plant cell such that the plant cellis visibly bioluminescent. A recombinant nucleic acid molecule of theinvention is exemplified by, in operative linkage, a CaMV 35S enhancer,a CaMV 35S promoter, a TEV translational enhancer, a nucleotide sequenceencoding a luciferase polypeptide or variant thereof, and a CaMV 35Sterminator. In one embodiment, the CaMV 35S enhancer comprises a dualCaMV 35S enhancer, and the luciferase polypeptide or variant thereof isa luc+luciferase variant. In another embodiment, a recombinant nucleicacid molecule of the invention is exemplified by the nucleotide sequenceset forth as about nucleotides 8366 to 11,113 of SEQ ID NO:2.

A recombinant nucleic acid molecule of the invention also is exemplifiedby the sequence set forth as nucleotides 8340 to 12,098 of SEQ ID NO:4,which includes, in operative linkage, a metallothionein gene (MT2B)regulatory element comprising an enhancer and promoter (nucleotides11,000 to 12,098 of SEQ ID NO:4), an omega translational enhancer(nucleotides 10,675 to 10,741 of SEQ ID NO:4), a nucleotide sequenceencoding a luciferase polyp eptide or variant thereof such as luc⁺(nucleotides 9015 to 10,667 or SEQ ID NO:4) and an RbcS E9 polyA region(nucleotides 8340 to 8981 of SEQ ID NO:4). In addition, a recombinantnucleic acid molecule of the invention also is exemplified by thesequence set forth as nucleotides 6 to 3570 of SEQ ID NO:5, whichincludes, in operative linkage, an actin 2 regulatory element comprisingan enhancer and promoter (nucleotides 6 to 1145 of SEQ ID NO:5), anomega translational enhancer (nucleotides 1169 to 1235 of SEQ ID NO:5),a nucleotide sequence encoding a luciferase polypeptide or variantthereof such as luc⁺ (nucleotides 1243 to 2895 of SEQ ID NO:5), and anRbcS E9 polyA region (nucleotides 2929 to 3570 of SEQ ID NO:5).

A recombinant nucleic acid molecule of the invention comprises anucleotide sequence encoding a bioluminescent polypeptide operativelylinked to one or more regulatory elements, for example, a translationalenhancer. As used herein, the term “operatively linked,” when used inreference to a regulatory element and a second nucleotide sequence,which can be another regulatory element or a coding or other sequence,means that the regulatory element is positioned with respect to thesecond nucleotide sequence such that the regulatory element effects itsfunction with respect to the second nucleotide sequence in substantiallythe same manner as it does when the regulatory element is present in itsnatural position in a genome. Transcriptional promoters, for example,generally act in a position and orientation dependent manner, andusually are positioned at or within about five nucleotides to aboutfifty nucleotides 5′ (upstream) of the start site of transcription of agene in nature. In comparison, enhancers can act in a relativelyposition or orientation independent manner, and can be positionedseveral hundred or thousand nucleotides upstream or downstream from atranscription start site, or in an intron within the coding region of agene, yet still be operatively linked to the coding region so as toenhance transcription. The relative positions and orientations ofvarious regulatory elements, including the positioning of a transcribedregulatory sequence such as an IRES or a translated regulatory elementsuch as a cell compartmentalization domain in an appropriate readingframe, are well known and methods for operatively linking such elementsare routine in the art (see, for example, Sambrook et al., “MolecularCloning: A laboratory manual” (Cold Spring Harbor Laboratory Press1989); Ausubel et al., “Current Protocols in Molecular Biology” (JohnWiley and Sons, Baltimore Md. 1987, and supplements through 1995), eachof which is incorporated herein by reference).

As used herein, the term “nucleic acid molecule” or “polynucleotide” or“nucleotide sequence” means a sequence of two or moredeoxyribonucleotides or ribonucleotides that are linked together by aphosphodiester bond. As such, the terms include RNA and DNA, which canbe a gene or a portion thereof, a cDNA, a synthetic polydeoxyribonucleicacid sequence, or the like, and can be single stranded or doublestranded, as well as a DNA/RNA hybrid. Furthermore, the terms are usedherein to include naturally occurring nucleic acid molecules, which canbe isolated from a cell, as well as synthetic molecules, which can beprepared, for example, by methods of chemical synthesis or by enzymaticmethods such as by the polymerase chain reaction (PCR). The term“recombinant” is used herein to refer to a nucleic acid molecule that isproduced by linking together at least two nucleotide sequences that arenot generally linked together in nature. As such, a recombinant nucleicacid molecule encompassed within the present invention isdistinguishable from a nucleic acid molecule that may be produced, forexample, during meiosis.

In general, the nucleotides comprising a recombinant nucleic acidmolecule, polynucleotide or nucleotide sequence are naturally occurringdeoxyribonucleotides, such as adenine, cytosine, guanine or thyminelinked to 2′-deoxyribose, or ribonucleotides such as adenine, cytosine,guanine or uracil linked to ribose. However, a nucleic acid molecule ornucleotide sequence also can contain nucleotide analogs, includingnon-naturally occurring synthetic nucleotides or modified naturallyoccurring nucleotides. Such nucleotide analogs are well known in the artand commercially available, as are polynucleotides containing suchnucleotide analogs (Lin et al., Nucl. Acids Res. 22:5220-5234, 1994;Jellinek et al., Biochemistry 34:11363-11372, 1995; Pagratis et al.,Nature Biotechnol. 15:68-73, 1997, each of which is incorporated hereinby reference).

Similarly, the covalent bond linking the nucleotides of a nucleotidesequence generally is a phosphodiester bond. However, the covalent bondalso can be any of numerous other bonds, including a thiodiester bond, aphosphorothioate bond, a peptide-like bond or any other bond known tothose in the art as useful for linking nucleotides to produce syntheticpolynucleotides (see, for example, Tam et al., Nucl. Acids Res.22:977-986, 1994; Ecker and Crooke, BioTechnology 13:351360, 1995, eachof which is incorporated herein by reference). The incorporation ofnon-naturally occurring nucleotide analogs or bonds linking thenucleotides or analogs can be particularly useful where the nucleic acidmolecule is to be exposed to an environment that can contain anucleolytic activity, including, for example, a plant tissue culturemedium or in a plant cell since the modified molecules can be lesssusceptible to degradation.

A nucleotide sequence containing naturally occurring nucleotides andphosphodiester bonds, can be chemically synthesized or can be producedusing recombinant DNA methods, using an appropriate polynucleotide as atemplate. In comparison, a nucleotide sequence containing nucleotideanalogs or covalent bonds other than phosphodiester bonds generally arechemically synthesized, although an enzyme such as T7 polymerase canincorporate certain types of nucleotide analogs into a polynucleotideand, therefore, can be used to produce such a polynucleotiderecombinantly from an appropriate template (Jellinek et al., supra,1995).

A recombinant nucleic acid molecule of the invention can be introducedinto a cell as a naked DNA molecule, can be incorporated in a matrixsuch as a liposome or a particle such as a viral particle, or can beincorporated into a vector. Incorporation of the polynucleotide into avector can facilitate manipulation of the polynucleotide, orintroduction of the polynucleotide into a plant cell. Accordingly, thepresent invention also provides a vector containing a recombinantnucleic acid comprising a nucleotide sequence encoding a bioluminescentpolypeptide operatively linked to one or more regulatory elements.

A vector can be derived from a plasmid or a viral vector such as a T-DNAvector (Horsch et al., Science 227:1229-1231 (1985), which isincorporated herein by reference). If desired, the vector can comprisecomponents of a plant transposable element, for example, a Ds transposon(Bancroft and Dean, Genetics 134:1221-1229, 1993, which is incorporatedherein by reference) or an Spm transposon (Aarts et al., Mol. Gen.Genet. 247:555-564, 1995, which is incorporated herein by reference). Inaddition to containing a recombinant nucleic acid molecule of theinvention, the vector also can contain various nucleotide sequences thatfacilitate, for example, rescue of the vector from a transformed plantcell; passage of the vector in a host cell, which can be a plant,animal, bacterial, or insect host cell; or expression of an encodingnucleotide sequence in the vector, including all or a portion of arescued coding region. As such, a vector can contain any of a number ofadditional transcription and translation elements, includingconstitutive and inducible promoters, enhancers, and the like (see, forexample, Bitter et al., Meth. Enzymol. 153:516-544, 1987). For example,a vector can contain elements useful for passage, growth or expressionin a bacterial system, including a bacterial origin of replication; apromoter, which can be an inducible promoter; and the like. A vectoralso can contain one or more restriction endonuclease recognition andcleavage sites, including, for example, a polylinker sequence, tofacilitate insertion or removal of a recombinant nucleic acid molecule.

In addition to a nucleotide sequence encoding a bioluminescentpolypeptide, a recombinant nucleic acid molecule of the invention, or avector containing such a recombinant nucleic acid molecule, can containone or more other expressible nucleotide sequences encoding an RNA or apolypeptide of interest. For example, the additional nucleotide sequencecan encode an antisense nucleic acid molecule; an enzyme such asβ-galactosidase, β-glucuronidase, luciferase, alkane phosphatase,glutathione S-transferase, chloramphenicol acetyltransferase, guaninexanthine phosphoribosyltransferase, and neomycin phosphotransferase; aviral polypeptide or a peptide portion thereof; or a growth factor or ahormone, which can be a plant growth factor or hormone. Expression ofsuch a nucleotide sequence can provide a means for selecting for a cellcontaining the construct, for example, by conferring a desirablephenotype to a plant cell containing the nucleotide sequence. Forexample, the additional nucleotide sequence can be, or encode, aselectable marker, which, when present or expressed in a plant cell,provides a means to identify the plant cell containing the marker. Aselectable marker provides a means for screening a population of plants,or plant cells, to identify those having the marker and, therefore, arecombinant nucleic acid molecule comprising a nucleotide sequenceencoding a bioluminescent polypeptide. A selectable marker generallyconfers a selective advantage to the plant cell, or a plant containingthe cell, for example, the ability to grow in the presence of a negativeselective agent such as an antibiotic or herbicide. A selectiveadvantage also can be due, for example, to an enhanced or novel capacityto utilize an added compound as a nutrient, growth factor or energysource. A selective advantage can be conferred by a singlepolynucleotide, or its expression product, or by a combination ofpolynucleotides whose expression in a plant cell gives the cell apositive selective advantage, a negative selective advantage, or both.It should be recognized that expression of a bioluminescent polypeptidealso provides a means to a select plant cell containing the encodingnucleotide sequence. However, the use of an additional selectablemarker, which, for example, allows a plant cell to survive underotherwise toxic conditions, provides a means to enrich for transformedplant cells containing the recombinant nucleic acid molecule.

Examples of selectable markers include those described above, as well asthose that confer antimetabolite resistance, for example, dihydrofolatereductase, which confers resistance to methotrexate (Reiss, PlantPhysiol. (Life Sci. Adv.) 13:143-149, 1994); neomycinphosphotransferase, which confers resistance to the aminoglycosidesneomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2:987-995,1983) and hygro, which confers resistance to hygromycin (Marsh, Gene32:481-485, 1984), trpB, which allows cells to utilize indole in placeof tryptophan; hisD, which allows cells to utilize histinol in place ofhistidine (Hartman, Proc. Natl. Acad. Sci., USA 85:8047, 1988);mannose-6-phosphate isomerase which allows cells to utilize mannose (WO94/20627); ornithine decarboxylase, which confers resistance to theornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine(DFMO; McConlogue, 1987, In: Current Communications in MolecularBiology, Cold Spring Harbor Laboratory ed.); and deaminase fromAspergillus terreus, which confers resistance to Blasticidin S (Tamura,Biosci. Biotechnol. Biochem. 59:2336-2338, 1995). Additional selectablemarkers include those that confer herbicide resistance, for example,phosphinothricin acetyltransferase gene, which confers resistance tophosphinothricin (White et al., Nucl. Acids Res. 18:1062, 1990; Spenceret al., Theor. Appl. Genet. 79:625-631, 1990), a mutant EPSPV-synthase,which confers glyphosate resistance (Hinchee et al., BioTechnology91:915-922, 1998), a mutant acetolactate synthase, which confersimidazolione or sulfonylurea resistance (Lee et al., EMBO J.7:1241-1248, 1988), a mutant psbA, which confers resistance to atrazine(Smeda et al., Plant Physiol. 103:911-917, 1993), or a mutantprotoporphyrinogen oxidase (see U.S. Pat. No. 5,767,373), or othermarkers conferring resistance to an herbicide such as glufosinate. Inaddition, markers that facilitate identification of a plant cellcontaining the polynucleotide encoding the marker include, for example,luciferase (Giacomin, Plant Sci. 116:59-72, 1996; Scikantha, J.Bacteriol. 178:121, 1996), green fluorescent protein (Gerdes, FEBS Lett.389:4447, 1996) or fl-glucuronidase (Jefferson, EMBO J. 6:3901-3907,1997), and numerous others as disclosed herein or otherwise known in theart. Such markers also can be used as reporter molecules.

As disclosed herein, a vector of the invention contains a recombinantnucleic acid molecule comprising one or more regulatory elements inoperative linkage with a nucleotide sequence encoding a bioluminescentpolypeptide. For example, the recombinant nucleic acid molecule in avector can contain, in operative linkage, a plant enhancer, plantpromoter, a translational enhancer, a nucleotide sequence encoding abioluminescent polypeptide, a translation termination sequence, andtranscription termnination sequence. It should be recognized that one ormore of the regulatory elements initially can be a component of thevector or of the recombinant nucleic acid molecule.

In one embodiment, a vector of the invention contains a recombinantnucleic acid molecule comprising, in operative linkage, a CaMV 35Senhancer, a CaMV 35S promoter, a TEV translational enhancer, anucleotide sequence encoding a luciferase polypeptide or variantthereof, and a CaMV 35S terminator. In another embodiment, therecombinant nucleic acid molecule contained in the vector comprises, inoperative linkage, a dual CaMV 35S enhancer, a CaMV 35S promoter, a TEVtranslational enhancer, a nucleotide sequence encoding a luc⁺ luciferasevariant, and a CaMV 35S terminator. Vectors of the invention areexemplified herein by a vector having the nucleotide sequence of SEQ IDNO:1 (see, also, FIG. 1), and by a vector having the nucleotide sequenceof SEQ ID NO:2 (see, also, FIG. 2). Additional vectors of the inventionare exemplified by those set forth as SEQ ID NO:4 and SEQ ID NO:5. Thepresent invention also provides a host cell containing a recombinantnucleic acid molecule of the invention, or containing a vector of theinvention.

The present invention further provides a method of producing agenetically modified plant cell that is visibly bioluminescent. As usedherein, the term “genetically modified,” when used in reference to aplant cell, means that the plant cell contains an exogenously introducednucleic acid molecule. For purposes of the present invention, theintroduced nucleotide sequence generally is a recombinant nucleic acidmolecule comprising one or more regulatory elements operatively linkedto a nucleotide sequence encoding a bioluminescent polypeptide, or avector containing such a recombinant nucleic acid molecule. The nucleicacid molecule can be transiently associated with the plant cell, butgenerally is stably associated with the genetically modified cell, aswell as its progeny, either as an autonomously replicating molecule ordue to integration into the plant cell genomic DNA or plastid DNA thatis stably maintained in the plant cell. If desired, a transgenecomprising a recombinant nucleic acid molecule of the invention can beintroduced into a plant genome in a site-specific matter, for example,by homologous recombination.

A method of the invention can be performed, for example, by introducinga transgene comprising a recombinant nucleic acid molecule of theinvention into a plant cell such that the encoded bioluminescentpolypeptide is expressed in the plant cell at a level that can produceat least about 750,000 to 1×10⁶ photons of visible light/mm²/second. Assuch, the present invention also provides a genetically modified plantcell produced by a method of the invention, as well as to a transgenicplant containing or generated from such a genetically modified plantcell, and to plant tissues, plant cells, plant organs and seeds producedby such a transgenic plant; as well as to a cDNA or genomic DNA libraryprepared from the transgenic plant, or from a plant cell or plant tissueobtained from the transgenic plant. A method of the invention canfurther include contacting the plant cell, or the genetically modifiedplant cell derived therefrom, with an agent that reduces or inhibitspigment synthesis in the plant cell or in a tissue or organ of atransgenic plant of the invention, for example, the synthesis of ananthocyanin, which is a water-soluble red to blue plant pigment; acarotene, which is an orange-yellow pigment located in the chloroplasts;chlorophyll, which is the green pigment in green plants; xanthophyll,which is a yellow to colorless photosynthetic plant pigment, or anyother pigment that can absorbs visible light, for example, lycopene,which is a red pigment found in tomatoes. The agent that is selected forreducing or inhibiting pigmentation of a plant is based on theparticular pigment, and that the pathway involved in synthesis of thepigment. For example, an agent such as norfluorazon, which inhibitscarotenoid synthesis, can be used to reduce the amount of chlorophyll inthe plant cell, thus enhancing the emission of a greater number ofphotons from the plant cell, or from a transgenic plant derived from theplant cell.

The term “plant” is used broadly herein to include any plant at anystage of development, or to part of a plant, including a plant cutting,a plant cell, a plant cell culture, a plant organ, a plant seed, and aplantlet. A plant cell is the structural and physiological unit of theplant, comprising a protoplast and a cell wall. A plant cell can be inthe form of an isolated single cell or aggregate of cells such as afriable callus, or a cultured cell, or can be part of higher organizedunit, for example, a plant tissue, plant organ, or plant. Thus, a plantcell can be a protoplast, a gamete producing cell, or a cell orcollection of cells that can regenerate into a whole plant. As such, aseed, which comprises multiple plant cells and is capable ofregenerating into a whole plant, is considered plant cell for purposesof this disclosure. A plant tissue or plant organ can be a seed,protoplast, callus, or any other groups of plant cells that is organizedinto a structural or functional unit. Particularly useful parts of aplant include harvestable parts and parts usefull for propagation ofprogeny plants. A harvestable part of a plant can be any useful part ofa plant, for example, flowers, pollen, seedlings, tubers, leaves, stems,fruit, seeds, roots, and the like. A part of a plant useful forpropagation includes, for example, seeds, fruits, cuttings, seedlings,tubers, rootstocks, and the like.

A transgenic plant can be regenerated from a genetically modified plantcell, i.e., a whole plant can be regenerated from a plant cell; a groupof plant cells; a protoplast; a seed; or a piece of a plant such as aleaf, a cotyledon or a cutting. Regeneration from protoplasts variesfrom species to species of plants. For example, a suspension ofprotoplasts can be made and, in certain species, embryo formation can beinduced from the protoplast suspension, to the stage of ripening andgermination. The culture media generally contains various componentsnecessary for growth and regeneration, including, for example, hormonessuch as auxins and cytokinins; and amino acids such as glutamic acid andproline, depending on the particular plant species. Efficientregeneration will depend, in part, on the medium, the genotype, and thehistory of the culture. If these variables are controlled, however,regeneration is reproducible.

Regeneration can occur from plant callus, explants, organs or plantparts. Transformation can be performed in the context of organ or plantpart regeneration. (see Meth. Enzymol. Vol. 118; Klee et al. Ann. Rev.Plant Physiol. 38:467 (1987), which is incorporated herein byreference). Utilizing the leaf disk-transformation-regeneration method,for example, disks are cultured on selective media, followed by shootformation in about two to four weeks (see Horsch et al., supra, 1985).Shoots that develop are excised from calli and transplanted toappropriate root-inducing selective medium. Rooted plantlets aretransplanted to soil as soon as possible after roots appear. Theplantlets can be repotted as required, until reaching maturity.

In vegetatively propagated crops, the mature tralsgenic plants arepropagated utilizing cuttings or tissue culture techniques to producemultiple identical plants. Selection of desirable transgenotes is madeand new varieties are obtained and propagated vegetatively forcommercial use. In seed propagated crops, the mature transgenic plantscan be self-pollinated to produce a homozygous inbred plant. Theresulting inbred plant produces seeds that contain the introducedtransgene, which can comprise a heterologous nucleotide sequenceencoding a bioluminescent polypeptide, and can be grown to produceplants that express the polypeptide. As such, the invention providesseeds produced by a transgenic plant obtained by a method of theinvention.

Transgenic plants comprising different transgenes also can be crossbred,thereby providing a means to obtain transgenic plants containing two ormore different transgenes, one of which encodes a bioluminescentpolypeptide and the other or others of which confer a desirablecharacteristic to the plant. Methods for breeding plants and selectingfor crossbred plants having desirable characteristics or othercharacteristics of interest are well known in the art.

A method of the invention can be performed by introducing a recombinantnucleic acid molecule of the invention into a plant cell. As usedherein, the term “introducing” means transferring a polynucleotide intoa plant cell. A nucleic acid molecule can be introduced into a cell by avariety of methods. For example, the recombinant nucleic acid moleculeof the invention, which can be contained in a vector, can be introducedinto a plant cell using a direct gene transfer method such aselectroporation or microprojectile mediated transformation, or usingAgrobacterium mediated transformation. As used herein, the term“transformed” refers to a plant cell containing an exogenouslyintroduced nucleic acid molecule.

It should be recognized that one or more nucleic acid molecules, whichare the same or different and at least one of which is a recombinantnucleic acid molecule of the invention, can be introduced into a plantcell, thereby providing a means to obtain a genetically modified plantcell containing multiple copies of a single transgene, or containing twoor more different genes, either or both of which can be present inmultiple copies. Such genetically modified plant cells can be produced,for example, by simply selecting plant cells having multiple copies of asingle type of transgene; by cotransfecting plant cells with two or morepopulations of different transgenes and identifying those containing thetwo or more different transgenes; or by crossbreeding transgenic plants,each of which contains one or more desired transgenes, and identifyingthose progeny having the desired transgenes.

Methods for introducing a nucleic acid molecule into a plant cell toobtain a transformed plant also include direct gene transfer (seeEuropean Patent A 164 575), injection, electroporation, biolisticmethods such as particle bombardment, pollen-mediated transformation,plant RNA virus-mediated transformation, liposome-mediatedtransformation, transformation using wounded or enzyme-degraded immatureembryos, or wounded or enzyme-degraded embryogenic callus, and the like.Transformation methods using Agrobacterium tumefaciens tumor inducing(Ti) plasmids or A. rhizogenes root-inducing (Ri) plasmids, or otherplant virus vectors are well known in the art (see, for example, WO99/47552; Weissbach and Weissbach, “Methods for Plant Molecular Biology”(Academic Press, NY 1988), section VIII, pages 421-463; Grierson andCorey, “Plant Molecular Biology” 2d Ed. (Blackie, London 1988), Chapters7-9, each of which is incorporated herein by reference; Horsch et al.,supra, 1985). The wild type form of A. tumefaciens, for example,contains a Ti plasmid, which directs production of tumorigenic crowngall growth on host plants. Transfer of the tumor inducing T-DNA regionof the Ti plasmid to a plant genome requires the Ti plasmid-encodedvirulence genes as well as T-DNA borders, which are a set of direct DNArepeats that delineate the region to be transferred. An Agrobacteriumbased vector is a modified form of a Ti plasmid, in which the tumorinducing functions are replaced by a nucleotide sequence of interestthat is to be introduced into the plant host.

Methods of using Agrobacterium mediated transformation includecocultivation of Agrobacterium with cultured isolated protoplasts;transformation of plant cells or tissues with Agrobacterium; andtransformation of seeds, apices or meristems with Agrobacterium. Inaddition, in planta transformation by Agrobacterium can be performedusing vacuum infiltration of a suspension of Agrobacterium cells(Bechtold et al., C.R. Acad. Sci. Paris 316:1194, 1993, which isincorporated herein by reference).

Agrobacterium mediated transformation can employ cointegrate vectors orbinary vector systems, in which the components of the Ti plasmid aredivided between a helper vector, which resides permanently in theAgrobacterium host and carries the virulence genes, and a shuttlevector, which contains the gene of interest bounded by T-DNA sequences.Binary vectors are well known in the art (see, for example, De Framond,BioTechnology 1:262, 1983; Hoekema et al., Nature 303:179, 1983, each ofwhich is incorporated herein by reference) and are commerciallyavailable (Clontech; Palo Alto Calif.). For transformation,Agrobacterium can be cocultured, for example, with plant cells orwounded tissue such as leaf tissue, root explants, hypocotyls,cotyledons, stem pieces or tubers (see, for example, Glick and Thompson,“Methods in Plant Molecular Biology and Biotechnology” (Boca Raton Fla.,CRC Press-1993), which is incorporated herein by reference). Woundedcells within the plant tissue that have been infected by Agrobacteriumcan develop organs de novo when cultured under the appropriateconditions; the resulting transgenic shoots eventually give rise totransgenic plants, which contain an exogenously introducedcircadian-regulated polynucleotide, or an oligonucleotide portionthereof.

Agrobacterium mediated transformation has been used to produce a varietyof tratsgenic plants, including, for example, transgenic cruciferousplants such as Arabidopsis, mustard, rapeseed and flax; transgenicleguminous plants such as alfalfa, pea, soybean, trefoil and whiteclover; and transgenic solanaceous plants such as eggplant, petunia,potato, tobacco and tomato (see, for example, Wang et al.,“Transformation of Plants and Soil Microorganisms” (Cambridge,University Press 1995), which is incorporated herein by reference). Inaddition, Agrobacterium mediated transformation can be used to introducean exogenous nucleic acid molecule into apple, aspen, belladonna, blackcurrant, carrot, celery, cotton, cucumber, grape, horseradish, lettuce,morning glory, muskmelon, neem, poplar, strawberry, sugar beet,sunflower, walnut, asparagus, rice and other plants (see, for example,Glick and Thompson, supra, 1993; Hiei et al., Plant J. 6:271-282, 1994;Shimamoto, Science 270:1772-1773, 1995).

Suitable strains of A. tumefaciens and vectors as well as transformationof Agrobacteria and appropriate growth and selection media are wellknown in the art (GV3101, pMK90RK), Koncz, Mol. Gen. Genet. 204:383-396,1986; (C58C1, pGV3850kan), Deblaere, Nucl. Acid Res. 13:4777, 1985;Bevan, Nucleic Acid Res. 12:8711, 1984; Koncz, Proc. Natl. Acad. Sci.USA 86:8467-8471, 1986; Koncz, Plant Mol. Biol. 20:963-976, 1992; Koncz,Specialized vectors for gene tagging and expression studies. In: PlantMolecular Biology Manual Vol. 2, Gelvin and Schilperoort (Eds.),Dordrecht, The Netherlands: Kluwer Academic Publ. (1994), 1-22; EuropeanPatent A-1 20 516; Hoekema: The Binary Plant Vector System,Offsetdruikkerij Kanters B. V., Alblasserdam (1985), Chapter V; Fraley,Crit. Rev. Plant. Sci., 4:1-46; An, EMBO J. 4:277-287, 1985).

Where the recombinant nucleic acid molecule is contained in a vector,the vector can contain functional elements, for example “left border”and “right border” sequences of the T-DNA of Agrobacterium, which allowfor stable integration into a plant genome. Furthermore, methods andvectors that permit the generation of marker-free transgenic plants, forexample, where a selectable marker gene is lost at a certain stage ofplant development or plant breeding, are known, and include, forexample, methods of co-transformation (Lyznik, Plant Mol. Biol.13:151-161, 1989; Peng, Plant Mol. Biol. 27:91-104, 1995), or methodsthat utilize enzymes capable of promoting homologous recombination inplants (see, e.g., W097/08331; Bayley, Plant Mol. Biol. 18:353-361,1992; Lloyd, Mol. Gen. Genet. 242:653-657, 1994; Maeser, Mol. Gen.Genet. 230:170-176, 1991; Onouchi, Nucl. Acids Res. 19:6373-6378, 1991;see, also, Sambrook et al., supra, 1989).

A direct gene transfer method such as electroporation also can be usedto introduce a polynucleotide into a cell such as a plant cell. Forexample, plant protoplasts can be electroporated in the presence of arecombinant nucleic acid molecule comprising a nucleotide sequenceencoding a bioluminescent polypeptide, which can be in a vector (Frommet al., Proc. Natl. Acad. Sci., USA 82:5824, 1985, which is incorporatedherein by reference). Electrical impulses of high field strengthreversibly permeabilize membranes allowing the introduction of thenucleic acid. Electroporated plant protoplasts reform the cell wall,divide and form a plant callus. Microinjection can be performed asdescribed in Potrykus and Spangenberg (eds.), Gene Transfer To Plants.Springer Verlag, Berlin, N.Y. (1995). A transformed plant cellcontaining the introduced recombinant nucleic acid molecule can beidentified due to the presence of a selectable marker included in theconstruct, or simply by looking at the plant cells and observing visiblebioluminescence.

Microprojectile mediated transformation also can be used to introduce arecombinant nucleic acid molecule of the invention into a plant cell(Klein et al., Nature 327:70-73, 1987, which is incorporated herein byreference). This method utilizes microprojectiles such as gold ortungsten, which are coated with the desired nucleic acid molecule byprecipitation with calcium chloride, spermidine or polyethylene glycol.The microprojectile particles are accelerated at high speed into a planttissue using a device such as the BIOLISTIC PD-1000 particle gun(BioRad; Hercules Calif.).

Microprojectile mediated delivery (“particle bombardment”) is especiallyuseful to transform plant cells that are difficult to transform orregenerate using other methods. Methods for the transformation usingbiolistic methods are well known (Wan, Plant Physiol. 104:37-48, 1984;Vasil, BioTechnology 11:1553-1558, 1993; Christou, Trends in PlantScience 1:423-431, 1996). Microprojectile mediated transformation hasbeen used, for example, to generate a variety of transgenic plantspecies, including cotton, tobacco, corn, hybrid poplar and papaya (seeGlick and Thompson, supra, 1993). Important cereal crops such as wheat,oat, barley, sorghum and rice also have been transformed usingmicroprojectile mediated delivery (Duan et al., Nature Biotech.14:494-498, 1996; Shimamoto, Curr. Opin. Biotech. 5:158-162, 1994). Arapid transformation regeneration system for the production oftransgenic plants such as a system that produces transgenic wheat in twoto three months (see European Patent No. EP 0709462A2, which isincorporated herein by reference) also can be useful for producing atransgenic plant according to a method of the invention, thus allowingmore rapid identification of gene functions. The transformation of mostdicotyledonous plants is possible with the methods described above.Transformation of monocotyledonous plants also can be transformed using,for example, biolistic methods as described above, protoplasttransformation, electroporation of partially permeabilized cells,introduction of DNA using glass fibers, Agrobacterium mediatedtransformation, and the like.

Plastid transformation also can be used to introduce a nucleic acidmolecule into a plant cell (U.S. Pat. Nos. 5,451,513, 5,545,817, and5,545,818; WO 95/16783; McBride et al., Proc. Natl. Acad. Sci., USA91:7301-7305, 1994). Chloroplast transformation involves introducingregions of cloned plastid DNA flanking a desired nucleotide sequence,for example, a selectable marker together with polynucleotide ofinterest into a suitable target tissue, using, for example, a biolisticor protoplast transformation method (e.g., calcium chloride or PEGmediated transformation). One to 1.5 kb flanking regions (“targetingsequences”) facilitate homologous recombination with the plastid genome,and allow the replacement or modification of specific regions of theplastome. Using this method, point mutations in the chloroplast 16S rRNAand rps12 genes, which confer resistance to spectinomycin andstreptomycin and can be utilized as selectable markers fortransformation (Svab et al., Proc. Natl. Acad. Sci., USA 87:8526-8530,1990; Staub and Maliga, Plant Cell 4:39-45, 1992), resulted in stablehomopiasmic transformants, at a frequency of approximately one per 100bombardments of target leaves. The presence of cloning sites betweenthese markers allowed creation of a plastid targeting vector forintroduction of foreign genes (Staub and Maliga, EMBO J. 12:601-606,1993). Substantial increases in transformation frequency are obtained byreplacement of the recessive rRNA or r-protein antibiotic resistancegenes with a dominant selectable marker, the bacterial aadA geneencoding the spectinomycin-detoxifying enzymeaminoglycoside-3′-adenyltransferase (Svab and Maliga, Proc. Natl. Acad.Sci., USA 90:913-917, 1993). Approximately 15 to 20 cell division cyclesfollowing transformation are generally required to reach a homoplastidicstate. Plastid expression, in which genes are inserted by homologousrecombination into all of the several thousand copies of the circularplastid genome present in each plant cell, takes advantage of theenormous copy number advantage over nuclear-expressed genes to permitexpression levels that can readily exceed 10% of the total soluble plantprotein.

Plants suitable for treatment according to a method of the invention soas to be visibly bioluminescent can be monocots or dicots and include,but are not limited to, maize, wheat, barley, rye, sweet potato, bean,pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish,spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin,hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach,nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple,avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane,sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton,alfalfa, rice, potato, eggplant, cucumber, Arabidopsis thaliana, andwoody plants such as coniferous and deciduous trees. Thus, a transgenicplant or genetically modified plant cell of the invention can be anangiosperm or gymnosperm.

Angiosperms are divided into two broad classes based on the number ofcotyledons, which are seed leaves that generally store or absorb food; amonocotyledonous angiosperm has a single cotyledon, and a dicotyledonousangiosperm has two cotyledons. Angiosperms produce a variety of usefulproducts including materials such as lumber, rubber, and paper; fiberssuch as cotton and linen; herbs and medicines such as quinine andvinblastine; ornamental flowers such as roses and, where included withinthe scope of the present invention, orchids; and foodstuffs such asgrains, oils, fruits and vegetables.

Angiosperms encompass a variety of flowering plants, including, forexample, cereal plants, leguminous plants, oilseed plants, hardwoodtrees, fruit-bearing plants and ornamental flowers, which generalclasses are not necessarily exclusive. Cereal plants, which produce anedible grain cereal, include, for example, corn, rice, wheat, barley,oat, rye, orchardgrass, guinea grass, sorghum and turfgrass. Leguminousplants include members of the pea family (Fabaceae) and produce acharacteristic fruit known as a legume. Examples of leguminous plantsinclude, for example, soybean, pea, chickpea, moth bean, broad bean,kidney bean, lima bean, lentil, cowpea, dry bean, and peanut, as well asalfalfa, birdsfoot trefoil, clover and sainfoin. Oilseed plants, whichhave seeds that are useful as a source of oil, include soybean,sunflower, rapeseed (canola) and cottonseed.

Angiosperms also include hardwood trees, which are perennial woodyplants that generally have a single stem (trunk). Examples of such treesinclude alder, ash, aspen, basswood (linden), beech, birch, cherry,cottonwood, eln, eucalyptus, hickory, locust, maple, oak, persimmon,poplar, sycamore, walnut, sequoia, and willow. Trees are useful, forexample, as a source of pulp, paper, structural material and fuel.

Angiosperms are fruit-bearing plants that produce a mature, ripenedovary, which generally contains seeds. A fruit can be suitable for humanor animal consumption or for collection of seeds to propagate thespecies. For example, hops are a member of the mulberry family that areprized for their flavoring in malt liquor. Fruit-bearing angiospermsalso include grape, orange, lemon, grapefruit, avocado, date, peach,cherry, olive, plum, coconut, apple and pear trees and blackberry,blueberry, raspberry, strawberry, pineapple, tomato, cucumber andeggplant plants. An ornamental flower is an angiosperm cultivated forits decorative flower. Examples of commercially important ornamentalflowers include rose, lily, tulip and chrysanthemum, snapdragon,camellia, carnation and petunia plants, and can include orchids.

The skilled artisan will recognize that the methods of the invention canbe practiced using these or other angiosperms, as desired, as well asgymnosperms, which do not produce seeds in a fruit. As such, the methodsof the invention can be used to prepare bioluminescent plants of variousfamilies, including, for example, Asteraceae-Aster family; Fabaceae-Peafamily; Rubiaceae-Madder family; Poaceae-Grass family;Euphorbiaceae-Spurge family; Lamiaceae-Mint family,Melastomataceae-Melastome family; Liliaceae-Lily family;Scrophulariaceae-Figwort family; Acanthaceae-Ancanthus family,Myaceae-Myrtle family, Cyperaceae-Sedge family; Ericaceae-Heath family,Apiaceae-Carrot family, Rosaceae-Rose family; Brassicaceae-Mustardfamily, Araceae-Arum family, Asclepiadaceae-Milkweed family;Arecaceae-Pahm family, Solanaceae-Potato family, Boraginaceae-Boragefamily; Gesneriaceae-Generiad family, Lauraceae-Laurel family, andBromeliaceae-Bromeliad family. In certain embodiments, the methods ofthe invention also can be used to prepare bioluminescent plants ofOrchidaceae-Orchid family, whereas in other embodiments, members of theorchid family are specifically excluded.

A recombinant nucleic acid molecule of the invention or a vectorcontaining the recombinant nucleic acid molecule provides a valuablereagent for identifying plant cells containing an introduced nucleicacid molecule. For example, the recombinant nucleic acid molecule, orvector containing the recombinant nucleic acid molecule, can include anucleotide sequence of interest. Following transformation of plant cellswith the recombinant nucleic acid molecule of the invention, whichcontains the nucleotide sequence of interest, visibly bioluminescentplant cells can be selected simply by looking at the plant cells, forexample, under a microscope, and picking out the ones that are visiblybioluminescent, thereby selecting plant cells that also contain thenucleotide sequence of interest. Thus, a recombinant nucleic acidmolecule of the invention is useful as a selectable marker, and providesa quick, efficient, and inexpensive means to identify a plant cellcontaining a nucleotide sequence of interest.

A nucleotide sequence of interest also can encode an immunogenicpolypeptide, for example, a viral or bacterial polypeptide, or a peptideportion thereof. A genetically modified plant cell, particularly atransgenic plant comprising such a plant cell, can be used as a sourceof a vaccine, wherein the individuals to be immunized merely ingest theplant, and a protective or palliative immune response is obtained. As anadditional advantage, problems generally associated with the preparationand purification of a vaccine, as well as storage and refrigerationproblems and the like are obviated. In view of these examples, it willbe recognized that genetically modified plant cells and transgenic plantcomprising such cells containing essentially any nucleotide sequence ofinterest can be prepared, and that the inclusion of a recombinantnucleic acid molecule of the invention provides a selectable marker thatfacilitates the identification of a transformed plant cell or plantcomprising such a cell that contains the nucleotide sequence of interest

A recombinant nucleic acid molecule of the invention, or a vectorcontaining such a nucleic acid molecule, also is useful for preparingtransgenic plants having great ornamental value. Visibly bioluminescentplants containing genetically modified plant cells have not previouslybeen described. As such, the transgenic plants of the invention, which“glow in the dark,” provide a novel ornamental plant. For example, atransgenic plant expressing a luciferase polypeptide or variant thereofcan be sprayed with water containing luciferin, or the transgenic plantor a cutting or other portion of the transgenic plant, for example, aninflorescence of the plant, can be dipped or placed in a solutioncontaining luciferin, whereupon the visible bioluminescence is produced.For example, flowers can be cut from a transgenic plant of the inventionand placed in water containing luciferin, wherein the luciferin is takenup and transported to the flower, thus providing a visiblybioluminescent flower. Generally, the amount of luciferin sufficient toinduce visible bioluminescence includes at least about 0.1 mM, andconcentrations from about 0.1 mM to 5 mM or more can be used. Inaddition, the solution containing luciferin can further include asurfactant, for example, a non-ionic detergent such as TRITON X-100detergent or NONIDET P40 detergent in a concentration of about 0.001% toabout 0.1%, thus facilitating entry of the luciferin into the plantcells. Significantly, luciferase-mediated bioluminescence continues forseveral hours following a single administration of luciferin and,therefore, there is no requirement that the plants be continuallytreated to maintain bioluminescence. Furthermore, the transgenic plantscan be selected based on their expression of variable levels of light,including plants that glow dimly, yet visibly, and those that glow verybrightly.

In addition to providing general ornamental value, the visiblybioluminescent transgenic plants of the invention also can provide apractical value. For example, visibly bioluminescent transgenic plantsthat grow as bushes, shrubs or trees can be arranged along a walkway ordriveway. Where such plants express luciferase or a variant thereof, forexample, the plants can be watered in the early evening with a solutioncontaining luciferin such that they illuminate the walkway or drivewayafter night falls. Thus, the transgenic plants of the invention providethe additional advantage they can be used to make an area safer byilluminating the area.

The present invention also relates to kit, which contains a geneticallymodified plant cell of the invention, or a derivative of the geneticallymodified plant cell, for example, a transgenic plant derived from thegenetically modified plant cell, or a cell, a tissue, or an organ ofsuch a transgenic plant. As such, a kit of the invention can contain oneor more flowers, bracts, leaves or other tissues or organs, which, uponcontact with luciferin, are visibly luminescent.

Accordingly, a kit of the invention can further include an amount ofluciferin sufficient for generating visible bioluminescence of thegenetically modified plant cell or the derivative of the geneticallymodified plant cell, and also can include a plurality of such amounts ofluciferin, thus allowing the generation of visible bioluminesce a numberof times, as desired. In addition, a kit of the invention can include anamount of a carotenoid inhibitor, for example, norfluorazon, sufficientfor reducing or inhibiting chlorophyll production in the geneticallymodified plant cell or the derivative of the genetically modified plantcell.

In another embodiment, a kit of the invention contains one or morecuttings, seeds, or other portion or derivative of a visiblybioluminescent plant of the invention such that a visibly luminescenttransgenic plant can be grown therefrom. As such, the kit also caninclude reagents for growing a transgenic plant from the cutting orseed, including, for example, a suitable plant food or other nutrientsource required for growth of the particular plant. In addition, the kitcan include an amount of a carotenoid inhibitor sufficient for reducingor inhibiting chlorophyll production in a transgenic plant grown fromthe cutting or seed; and/or can include an amount of luciferinsufficient for generating visible bioluminescence of a transgenic plantgrown from the cutting or seed.

Generally, a kit of the invention includes a container for thecomponents of the kit. It should be recognized, however, that thecontainer can be any convenient means for holding a component of thekit, including, for example, a packet or collection of packetscontaining seeds obtained from a transgenic plant of the invention, orcontaining predetermined amounts of a reagent such as luciferin, whichcan be added to a specified amount of water to provide a solutionsufficient to generate visible bioluminescence from a composition of theinvention. In one embodiment, the kit comprises a flower cutting of atransgenic plant of the invention in a vase, and can further include oneor more predetermined amounts of a reagent such as luciferin. In anotherembodiment, the kit comprises a seed or a cutting obtained from atransgenic plant of the invention, and further includes a reagent suchas luciferin and/or norfluorazon, in which the seed or cutting, or aplant derived therefrom, can be grown.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE 1 Preparation of Luciferase Expression Vectors

This example provides a method for preparing luciferase constructs thatcan be incorporated into an expression vector and are useful forproducing a visibly bioluminescent plant.

Plasmid pRTL2, which contains a CaMV 35S promoter with duplicatedenhancer, a TEV leader and initial coding sequence from pTL-7SN, and aCaMV 35S polyadenylation (poly A) signal sequence (Topfer et al., Nucl.Acids Res. 15:5890, 1987; Restrepo et al., Plant Cell 2:987, 1990, eachof which is incorporated herein by reference; see, also, Carrington andFreed, supra, 1990) was sequentially digested with Nco I and Xba I, thenseparated by electrophoresis on a 0.8% agarose gel. The nucleotidesequence of the CaMV 35S promoter with duplicated enhancer, TEV leaderand initial coding sequence, and CaMV 35S poly A signal sequence ofpRTL2 is shown as SEQ ID NO:3. A DNA band of approximately 3.9 kbrepresenting the linear pRTL2 (see nucleotides 1 to 1188 and 2844 to5475 of SEQ ID NO:1) was isolated and extracted from the gel using aQIAQUICK DNA gel extraction kit (Qiagen; Valencia Calif.) according tomanufacturer's instructions. The Nco I site in the TEV leader containsthe start codon for translation. Translational fusions containing theinitial 20 amino acid residues of the TEV polyprotein are made byligation to one of the polylinker sites, provided care is taken to avoidthe STOP codon in the Xba I site (see SEQ ID NO:3).

In a parallel reaction, pGL3 (Promega Corp., Madison Wis.), whichcontains a modified luciferase coding sequence, luc⁺ (Groskreutz et al.,Promega Notes 50:2, 1995; U.S. Pat. No.5,670,356, each of which isincorporated herein by reference) was digested sequentially with Xba Iand Nco I, then separated by electrophoresis on a 0.8% agarose gel. Twobands were present—a 1.6 kb band representing the luc⁺ coding sequence(see U.S. Pat. No.5,670,356) and an approximately 3 kb band representingthe remainder of the pGL3 plasmid. The approximately 1.6 kb luc⁺ band(see nucleotides 1188 to 2844of SEQ ID NO:1) was isolated and extractedas described above.

The isolated luc⁺ sequence (4 μl) was ligated into the linearized pRTL2vector (1 μl) using ligase and ligation buffer from BRL (GaithersburgMd.). The ligation was performed overnight at 16° C. E. coli DH5α cellswere transformed using a BioRad Electroporator in a ratio of 2 μl ofligated DNA to 20 μl of cells. Following electroporation, thetransformed cells were incubated in LB medium for 1 hr at 37° C., thenwere plated on LB plates containing 50 μg/ml ampicillin. Ofapproximately 3000 colonies, 32 were selected and screened by PCR forthe luc⁺ insert. PCR primers were specific for the 5′ end of the TEVpromoter in pRTL2 and for the 3′ end of the poly A terminator in pRTL2(see SEQ ID NO:3). PCR analysis revealed that 30 of the coloniescontained the luc⁺ insert. The presence of the insert in 6 clones wasverified by restriction analysis, and two candidate clones, designatedTK1-7 and TK1-8, were sequenced to confirm the presence of the luc⁺insert.

The TK1-8 clone was digested at 37° C. for 2 hr with Hind III, and thefragment containing the CaMV 35S promoter with duplicated enhancer, TEVleader, luc⁺ coding sequence and poly A termination signal (designated“TK1829”; see FIG. 2, and nucleotides 8366 to 11,113 of SEQ ID NO:2) wasisolated by electrophoresis as described above. The binary vector,pPZP221, also was digested with Hind III and isolated. The linearizedpPZP221 vector was treated with shrimp alkaline phosphatase for 1 hr at37° C., to remove terminal phosphate groups, followed by 15 min at 65°C. to heat inactivate the enzyme, then the TK1829 insert and linearizedpPZP221 vector were ligated and transformed into DH5α cells as describedabove. Cells were plated on LB agar plates containing X-gal, IPTG, and100 μg/ml spectinomycin.

Forty-six white colonies were selected, and the presence of the luc⁺coding sequence was confirmed by PCR as described above; all werepositive for the luc⁺ sequence. Six samples were selected and theorientation of the insert was determined by restriction digest analysis,using Sca I or Nar I. One construct (#29) having the correct orientationwas selected. The binary vector containing the luc⁺ insert wasdesignated pPZPTK1829 (FIG. 2; SEQ ID NO:2), and the inserted expressionconstruct was designated TK1829 (nucleotides 8366 to 11,113 of SEQ IDNO:2). TK1829 was sequenced using primers specific for 5′ and 3′ insertflanking regions of the pPZP221 vector. The pRTL2 vector from which theTK1829 insert was obtained was designated pTK1829 (see FIG. 1; see,also, SEQ ID NO:1).

The pMT-OM-LUC⁺ (SEQ ID NO:4; FIG. 3) and pACT-OM-LUC⁺ (SEQ ID NO:5;FIG. 4) binary vector plasmids were prepared from the pPZP221 binaryvector, as described above. The metallothionein (MT) or actin (ACT)promoter was inserted immediately preceding the omega enhancer in a BamHI site, which provides a convenient site to substitute othertranscriptional regulatory elements.

EXAMPLE 2 Preparation and Characterization of Visibly BioluminescentPlants

This example provides a method for introducing a luciferase expressionconstruct into plants such that a genetically modified visiblybioluminescent plant is produced, and a method for determining athreshold value of photon emission required for visible luminescence.

A. Preparation of TK1829 Transformed Plants

The TK1829 expression construct was introduced from pPZPTK1829 intoAgrobacterium l strain ABI 1 with the help of pRK2013 by tri-parentalmating. Agrobacterium containing the TK1829 construct were selectedbased on their resistance to the 100 μg/ml spectinomycin, 50 μg/mlkanamycin, and 25 μg/ml chloramphenicol. TK1829-Agrobacterium strainswere grown, then DNA was purified and checked by restriction digestanalysis using Sca I (2 hr; 37° C.) to verify the presence of theinsert.

Briefly, TK1829-Agrobacterium was grown as described below, and 28 dayold plants were infiltrated. Supernova (Snv) strain plants and Columbia(wild type) plants were each infiltrated. Two sets of infiltrations wereperformed on Columbia and Snv plants. Each time, two 4 inch pots of eachline were used and each pot contained approximately 50 plants, for atotal of 200 plants per line infiltrated.

For in planta plant transformation, 4 inch pots were prepared withScott's Metro mix 200 soil and covered with window screen mesh. Soil waswet and mounded up, and the screen mesh was secured by wrapping a rubberband around the neck of the pot, maling sure that the mesh was incontact with the soil surface so that germinating seedlings could getthrough the mesh. Seeds were stratified in 10 to 50 ml of sterile waterand stored at 4° C. for 3 days prior to being applied to pots. Seedswere sown using a dropper, to spread seeds evenly over the surface ofthe mesh. Plants were grown at approximately 22° C., in 12 hr light:12hr dark per day to a stage at which bolts were anywhere from 3 to 10inches high. The bolts were cut off to induce growth of secondaryinflorescence.

Infiltration was most effective 4 to 5 days after decapitation of bolts,but can be performed up to 8 days later. For infiltration, 5 to 20 mlliquid preculture of Agrobactenium containing the appropriate constructwere grown for about 2 days in a shaking incubator at 28° C., then 400ml of LB was inoculated with the preculture and incubate for oneadditional day. The large culture generally was prepared the day beforethe infiltration was performed.

Transformed Agrobacterium cells were grown to an optical density at 600nm (OD600) of approximately 1.2, then harvested by centrifugation (4000rpm, 10 min, room temperature), and resuspended in infiltration medium(1/2×Murashige and Skoog salts; 1×Gamborg's vitamins; 5.0% sucrose; 0.44μM benzylamino-purine (10 μg per liter of a 1 ng/ml stock); pH adjustedto 5.7 using KOH; then add 200 μl/l Silwet; media need not be sterilizedif used immediately) to an OD 600 of approximately 0.8. The OD600 of anovernight culture generally was about 1.2 to 1.6 units. A 400 mlsuspension generally was adequate for an infiltration procedure. Thesame suspension can be used as many as three times.

About 150-200 ml of the Agrobacterium suspension was added to either abeaker or dish and the prepared plants were inverted into thesuspension. The plants were entirely submerged and kept in the solutionfor 10 to 15 min. The pots then were removed from the suspension anddrained thoroughly, laid on their side in a plastic flat, and coveredwith a plastic dome or Saran Wrap to maintain humidity. The flat wasuncovered the next day and the pots set upright. A little water wasadded to the bottom of the tray to help keep the humidity level highwhile the plants were recovering. The plants were grown for 3 to 4weeks, keeping the bolts from each pot together and separated fromneighboring pots and different infiltrations. Once plants began toyellow and senesce, watering was reduced or discontinued. Seeds wereharvested together from one pot.

Transformants were selected on 150×15mm selection plates (1/2×Murashigeand Skoog salts; 1×Gamborg's vitamins; 1% sucrose; 0.5 g/l MES; 0.8%agarose; pH to 5.7; autoclaved, then add 75 μg/ml gentamycin, 500 μg/mlcarbenicilin, 50 μg/ml kanamycin). Seeds were surface sterilize for 2min in 500 μl 70% ethanol; followed by 10 min in 40% bleach, 1% SDS, 59%water, then rinsed 3 times with sterile water. The seeds wereresuspended in 3 to 4 ml of sterile water and plated by pouring onto aplate and shaking until evenly distributed. The plates were allowed tosit for 5 to 10 min, then excess liquid was removed. Approximately 70 to100 μl of dry seeds were added per plate. Plants were allowed tostratify at 4° C. for 3 days, then transferred to a growth chamber.

In some experiments, the herbicide, norflurazon (5 μM;4-chloro-5-(methylamino)-2-{3-(tri-fluoromethyl)phenyl}-3(2H)-pyridazinone)was included in the growth medium. Norflurazon inhibits chlorophyllsynthesis, resulting in the growth of “white” plants. Treatment ofplants with norflurazon resulted in greater emission of visiblebioluminescence due to the lack of chlorophyll, which absorbs light andblocks light emission (see Tables).

After 7 to 10 days (depending on the antibiotic resistance selectedfor), transformants were identified as dark green plants with elongatedroots that penetrated the agar. These seedlings were transferred onto asecond set of selection plates. After several days, plants had 4 to 8true leaves and branching roots. Pseudo-resistant plants were unable tosurvive in this medium. The plants then were transferred to soil, andkept covered for the first few days.

Plants were allowed to recover, bolt, set seed and senesce. Seeds wereharvested when dry, then were further dried in a dessicator for 1 week.Yield was approximately 0.5 to 0.75 ml of seed per pot. T1 (transgenicfirst generation) seeds in the Columbia background were screened on 75μg/ml gentamycin and 500 μg/ml carbenicilin MS plates. T1 seeds in theSnv background were screened on 75 μg/ml gentamycin, 500 μg/mlcarbenicilin, and 50 μg/ml kanamycin (Snv strain contains a transgeneconferring kanamycin resistance).

Over an 8 week period, 1.6 ml (approximately 32,000 seeds) of each T1seed set was sterilized and planted in 200 μl (4000 seeds) aliquots on50 ml plates as described above. Plants were stratified for 3 days at 4°C., then moved to a growth chamber set for 12 hr light: 12 hr dark andapproximately 27° C. Sixty-seven T1 seedlings in the Snv background and25 T1 seedlings in the Columbia background grew under the selectionconditions, and were recovered and transferred to soil to harvest T2seed. It took approximately 12 to 16 weeks to take T1 seed to T2 seed,and varied between plants.

B. Characterization of TK1829 Transformed Plants

T2 seeds of Columbia and Supernova were sterilized and plated as above,and screened for antibiotic resistance. In addition, the seedlings werescreened for bioluminescence, visually and with the Hamamatsu photoncounting camera. T2 seedlings within individual families were selectedbased on their intensity and brightness. Three lines in the Snvbackground (Snv 11, Snv 37 and Snv 57) and two lines in the Columbiabackground (Col 10 and Col 19) were selected as the most visiblybioluminescent plants. Seventy-eight T3 individuals of Snv 11, which wasextremely bright, were selected based on their visible bioluminescence.Three of these (Snv 11-8, Snv 11-48, Snv 11-52) were selected as thebrightest and were maintained as individual lines.

For characterization of luciferase expression, tissue was collected from22 day old plants grown under the same conditions. Examination ofColumbia wild-type plants, Columbia plants transformed with 35S:Luc(“35S:Luc”) and Snv plants transformed with cab2:Luc (“Supernova”; seeHicks et al., supra, 1996) was included for comparison. In variousassays (see below), protein concentration was determined, relative lightunits (RLU) were acquired using a luminometer, western blot analysis wasperformed using recombinant luciferase antibodies, plants were imagedusing a Night Owl Camera (see below) to obtain counts of light/mm²/sec,and plants were inspected visually to confirm that bioluminescence wasvisible to the eye. Based on these assays, the approximate amounts ofluciferase per microgram of total protein and the fold increase inenzyme activity were determined, and the amount of light emitted by theplants was determined.

For protein assays, a few hundred 22 day old seedlings from plants grownunder the same conditions were placed in liquid nitrogen, ground into afine power, and stored in aliquots of approximately 200 μl volume on dryice or at −80° C. until the time proteins were extracted. Approximately100 mg of the powdered tissue was extracted in 500 μl of 1×Promega CellLysis Reagent (CLR), ground briefly with a mortar and pestle, thencentrifuged at 4° C. for 5 min 14,000 rpm. The supernatant was dividedinto 100 μl aliquots and transferred immediately to dry ice. Any samplesnot used within 1 hr were stored at −80° C. to preserve enzyme activity.Protein concentrations were determined for 20 μl of tissue extract usingthe DC Protein Assay Reagent (BioRad); optical density (OD) wasdetermined at 750 nm using a Pharmacia Ultrospec III spectrophotometer.

Acquired Relative Light Units (RLU) were obtained using a Turner TD20ELuminometer. Samples from plants containing the TK1829 construct werediluted 1/100 (5 μl/500 μl) in order for the reading to be within therange of the luminometer. Five μl of sample and 50 μl of PromegaLuciferase Assay Reagent (LAR) was used. The maximum value of the rangeof readings taken every 30 sec for the first 5 min of the reaction wasentered into the data tables. The amount of luciferase was approximatedby western blot analysis (Tables 1 and 2) or using the RLU data obtainedby luminometry and the total protein concentration obtained byspectrophotometry (Table 4).

Western blot analysis of the extracts was performed using enhancedchemifluoresence (ECF; Amersham) to visualize protein bands. For samplescontaining the TK1829 construct, 0.1 μg of total protein was used. Forall other samples, 1.0 μg of total protein was used. The 10-folddifference was necessary to keep the luciferase protein concentrationswithin the range of the assay. A luciferase primary antibody and analkaline phosphatase secondary antibody (Promega) were used to identifyprotein bands. Proteins were separated by electrophoresis on, 10% SDSPAGE gels (BioRad), then transferred to Hybond nitrocellulose. Imageswere visualized using a Molecular Dynamics Fluorimeter, and data wasanalyzed using ImageQuant software.

Luciferase was expressed in 35S:Luc transformed Columbia plants(“35S:Luc”) and cab2:Luc transformed Supernova plants (“Supernova”;Table 1), but these plants were not visibly bioluminescent when examinedby eye. In comparison, the TK1829 transformed Columbia plants werevisibly bioluminescent. These results indicate that the threshold forvisible bioluminescence is between about 85 and 125 pg luc⁺ protein/μgprotein as determined by western blot analysis (Table 1).

The TK1829 transformed Supernova plants expressed about 6.5-fold moreluc⁺ than the TK1829 transformed Columbia plants as determined bywestern blot analysis (Table 1). In addition, the Snv-TK1829 plantsemitted about 7-fold more photons of light/mm²/sec than the Col-TK1829plants. These results demonstrate that visible bioluminescence can beproduced in various Arabidopsis strains. In addition, growth of theTK1829 transformed Supernova plants in norflurazon resulted in an almost2-fold increase in luciferase protein as measured by luminometry, and agreater than 7-fold amount of light emitted as measured using the NightOwl imaging system.

This result demonstrates that the amount of visible bioluminescence canbe increased by inhibiting chlorophyll synthesis in the transgenicplants. TABLE 1 pg Luciferase Plant Line protein/μg total protein a)Columbia Wild Type 0.01 b) 35S:Luc transformed Columbia 7.0 c) cab2:Luctransformed Supernova 85.0 d) TK1829 transformed Columbia 125.0 e)TK1829 transformed Supernova 834.0 f) TK1829 transformed Supernova with1456.0 Norflurazon

A comparison of western blot data on various plant extracts allowed adetermination of the relative difference in luciferase proteinconcentration among the various plant lines (Table 2). Western blotanalysis confirmed that Arabidopsis transformed with the TK1829construct expressed a much greater amount of luciferase expression ascompared to plants transformed with a 35S:Luc construct (see Table 2band d) and as compared to cab2:Luc-transformed Snv (“Supernova”; seeTable 2, c and e).

The various plant lines also were examined by imaging analysis using theEGG Berthold Night Owl imaging system (see, also, Example 2C, below).Plants were sprayed with approximately 1 ml of 5 mM luciferin in asolution of 0.001% TRITON X-100 detergent. The camera settings were keptconsistent for all plant lines. However, the time duration for takingthe images was varied to accommodate the different levels ofbioluminescence in the various plant lines. All data was processed andaveraged for photon counts of light/area over the time the image wastaken (mm/sec); variation between individual plants was eliminated byanalyzing pools of plants. TABLE 2 Fold increase in luciferase PlantComparison protein concentration a) Supernova vs. 35S:Luc 5 b) TK1829transformed Columbia 128 vs. 35S:Luc c) TK1829 transformed Columbia vs.31 Supernova d) TK1829 transformed Supernova vs. 244 35S:Luc e) TK1829transformed Supernova vs. 53 Supernova f) TK1829 transformed Supernovawith 2 Norflurazon vs. the same line without g) TK1829 transformedSupernova with 450 Norflurazon vs. 35S:Luc

In parallel with the levels of luc⁺ expression as disclosed above, lightemission by the TK1829 transformed plants was substantially greater thanthat of Columbia plants transformed with the 35S:Luc construct(“35S:Luc”) or of Snv plants transformed with a cab2:Luc construct(“Supernova”; see Table 3). Light emission of Arabidopsis plantstransformed with TK1829, as measured by photon counts per area persecond, was increased by as much as 5,200 fold over 35S:Luc transformedColumbia plants (see Table 3g).

Plants also were inspected by eye to confirm that they were visiblybioluminescent. The TK1829 transformed Columbia and TK1829-transformedSupernova plants were visibly bioluminescent (whether grown in thepresence or absence of norflurazon), whereas the none of the otherplants, including the 35S:Luc transformed Columbia and cab2:Luctransformed Supernova plants, were visibly bioluminescent. These resultsdemonstrate that transformation of a plant with a construct that canexpress about 125 pg luc⁺/μg protein as determined by western blotanalysis can effectively generate visibly bioluminescent plants. TABLE 3Fold increase in light Plant Comparison (cts/mm²/sec) a) Supernova vs.35S:Luc 70 b) Tk1829 transformed Columbia vs. 35S:Luc 106 c) Tk1829transformed Columbia vs. 1.5 Supernova d) Tk1829 transformed Supernovavs. 712 35S:Luc e) TK1829 transformed Supernova vs. 10 Supernova f)TK1829 transformed Supernova with 7 Norflurazon vs. Supernova g) TK1829transformed Supernova with 5,200 Norflurazon vs. 35S:LucC. Threshold Photon Emission for Visible Bioluminescence

In order to provide a standard for determining the threshold number ofvisible light photons required for visible bioluminescence, the numberof photons of visible light emitted per square millimeter per second wasdetermined using a defined system. A Night Owl camera with WinLightProgram LB981 (EG&G Berthold; Bad Wildbad, Germany) was used, and allimages were acquired under the following conditions: Cosmic Suppression,ON; Defect Correction, ON; Star Killer, OFF; Camera Readout, SLOW; Gain,HIGH; Look up Table set to GAMMA. Measurements were taken in aphotography dark room with a plate-to-lens distance of 275 mm (standardsetting on Night Owl camera). Each plant was imaged for a timeappropriate to the amount of bioluminescence emitted and values werenormalized to photon counts/area/second.

Approximate photons/mm²/sec were calculated using a conversion ofdischarges per minute divided by counts per minute. The Night Owl camerasystem was calibrated using manufacturer-provided standards from theBeckman LS6500 scintillation system. Two standards were used. The firststandard contained scintillant alone, which represents background levelsof discharges per minute (dpm). The second standard contained an amountof carbon-14 in scintillant that produces 100,600 dpm. Images were takenusing 1.1 pixel binning with background subtraction, and values ofcounts per second (cps) were acquired. The cps then was multiplied by 60to obtain counts per minute (cpm). Each discharge is representative ofthe energy released by a disintegrating beta particle in the form of aphoton. “Dpm” divided by “cpm” then provided an approximate efficiencyfor the camera, which was used for all data values to estimate actualphotons emitted. For example, where the carbon-14 standard=3.6 cps andscintillant alone=2.2 cps, the difference=1.4 cps (actual cps)=84 cpm;and 100,600 dpm divided by 84 cpm=1,198 discharges/count(photons/count), which is used as a conversion factor to calculate thenumber of photons emitted by plant and detected by the camera Using theabove conditions and calculation, various wild type and transformedplants were examined and photons/mm²/sec were determined. No visiblydetectable bioluminescence was observed in the Columbia wt plants, the35S LUC-transformed plants or Supernova plants, whereas all of theTK1829 transformed plants were visibly bioluminescent. As shown in Table4, a threshold value of visible bioluminescence occurs between about700,000 photons/mm²/sec and 1×10⁶ photons/mm²/sec (compare Tables 4c and4d; corresponding to 126.4 and 1562.4 pg luc⁺/μg protein, respectively,as determined by luminometry and spectrophotometry; see Example 2B).TABLE 4 Photon Emission Plant Comparison (cts/mm²/sec) a) Columbiawild-type 16.8 b) 35S:Luc 10,063.0 c) Supernova 701,669.0 d)Tk1829-19-transformed Columbia 1,061,668.0 e) TK1829-11-transformedSupernova 7,160,446.0 f) TK1829-11-transformed Supernova 52,604,180.0with Norflurazon

These results demonstrate that the amount of light produced by thevarious transformed plants correlates with the amount of luciferaseexpressed in the plants, and establish standard conditions defining athreshold level of light emission required for visible bioluminescence.

These results further provide guidelines for generating otherconstructs, which contain various combinations of regulatory elements,that can be examined for the ability to confer visible bioluminescenceon a plant. For example, by comparing a plant transformed with arecombinant nucleic acid molecule comprising a nucleotide sequenceencoding a bioluminescent polypeptide operatively linked to one or moreregulatory elements, with plants transformed with a 35S:Luc construct ora cab2:Luc construct, recombinant nucleic acid molecules encompassedwithin the present invention and useful for generating visiblybioluminescent plant cells or transgenic plants comprising such cellscan be obtained. More directly, by transforming plants with variousconstructs and determining photon emission under standardized conditionsas described above, visibly bioluminescent plants can be obtained.

Furthermore, the results disclosed herein indicate that plant cellsother than Arabidopsis plant cells can be genetically modified using,for example, a TK1829 recombinant nucleic acid molecule, thus providinga means to obtain different species, strains and varieties of visiblybioluminescent transgenic plants. In this respect, genetically modifiedpetunia plants that exhibit exceptional bioluminescence also have beengenerated using the disclosed compositions and methods.

D. Preparation and Characterization of Bioluminescent Petunia Plants

pMT-OM-LUC⁺ or pACT-OM-LUC⁺ expression constructs were introduced fromMT-OM-LUC⁺ or pACT-OM-LUC⁺ into Agrobacterium strain ABI 1 and in plantatransformation of Arabidopsis and of petunia plants were performed asdescribed above.

Whole leaves from 10 week old petunia plants were removed and washed inwarm soapy water. Leaves were sterilized in 10% (v/v)chlorine for 15 minand rinsed 4 times with sterile water. Squares of approximately 1 cmwere cut from the leaves under sterile conditions with an scalpel andcultured for one day in pre-culture medium (MS medium supplemented with1 mg/l of 6-benzylaminopurine (BAP) and 0.1 μg/ml of alpha-naphthaleneacetic acid (NAA).

A. tumefaciens strain ABI carrying one of the plasmids was used toinoculate the pieces of leaves. Bacteria from a single colony were grownat 28° C. overnight in liquid LB medium on a rotary shaker (250 rpm).The medium was supplemented with 50 μg/ml kanamycin, 100 μg/mlspectinomycin and 34 μg/ml chloramphenicol. The next day, a 1:10dilution was made into LB medium (without antibiotics) and sub-culturedcells were grown for 4 to 5 hr. Cultures were diluted to an OD (660 nm)of 0.05 to 0.07 with pre-culture liquid medium.

Explants were inoculated with a log phase solution of Agrobacteriumadjusted to approximately 5×10⁸ cells. The leaf pieces were allow tosoak for 5 min in the bacteria solution, blotted on sterile Whatmanfilter discs to remove excess of solution, then replaced in the originalpre-culture plates and incubated for 2 days at 23° C. under low light.The leaf squares were transferred into shoot proliferation medium(pre-culture medium containing 75 μg/ml gentamycin and 500 μg/mlcarbenicillin). After 4 weeks, shoots were excised and placed on rootingmedium (MS medium supplemented with 60 μg/ml gentamycin and 500 μg/mlcarbenicillin). After approximately 5 weeks, the plants were transferredto soil.

As shown in Table 5, Arabidopsis plants containing the MT-OM-LUC⁺construct (designated “31” in Table 5) or the ACT-OM-LUC⁺ construct(“#27” and “#17”) were visibly biolunminescent (i.e., greater than750,000 photons of light/mm²/sec (column labeled “photons”). Theseresults demonstrate that the methods of the invention can be used tomake various constructs, which, when introduced into plant cells, resultin the generation of visibly bioluminescent plants.

Similarly, introduction of the TK 1829 construct or the ACT-OM-LUC⁺construct into petunia plants resulted in the generation ofbioluminescent petunia plants. Although petunia plants containing theMr-OM-LUC⁺ construct were not examined for photon counts, the meanRLU/μg was similar to those for the TK 1828 and ACT-OM-LUC⁺ transformedpetunias and the plants were visibly bioluminescent to the eye.

These results demonstrate that the constructs of the invention areuseful for generating a variety of visibly bioluminescent plants.Together, the results of these studies demonstrate the broad utility ofthe claimed methods for generating visibly bioluminescent plants.

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims. TABLE 5 21 dayold Weight OD OD conc. mean pgLuc/ plants ug (750 nm) (750 nm) (ug/ul)RLU 1 RLU 2 RLU/ug ug cts/mm²/sec photons MT Line 31902 31 99.3 0.0860.086 0.0238844 128.1 132.8 1092.34395 15.24 2408.4 2,885,263.20 ActinLines Actin 2-7 Col 31902 #27 98.9 0.047 0.046 0.0111278 38.37 39.6700.68 9.80 911.64 1,092,144.72 Actin 1-1 Col 31902 #17 98.2 0.109 0.1090.0313123 318.4 331.5 2,075.54 28.81 3342.4 4,004,195.20 Petunia ActinLine T3 102 0.062 0.065 5.0861595 31.46 30.96 1.23 0.02 840 1,006,320.00TK1829 Line T5 96 0.105 101 84.933336 525.9 520.1 1.23 0.02 3385.74,056,068.60 MT Line T3 90 0.106 0.105 3.6981111 22.82 20.23 1.16 0.02n/a control 27 0.089 0.09 0.0150032 0.028 0.021 0.33 0.00 16.3519,587.30

1. A genetically modified plant cell, comprising a heterologousnucleotide sequence encoding a bioluminescent polypeptide, wherein thebioluminescent polypeptide can be expressed in amount sufficient toproduce at least 750,000 photons of visible light/mm²/second.
 2. Thegenetically modified plant cell of claim 1, wherein the bioluminescentpolypeptide is luciferase.
 3. The genetically modified plant cell ofclaim 1, wherein the bioluminescent polypeptide is a luciferase variant.4. The genetically modified plant cell of claim 3, wherein theluciferase variant is a luc⁺ luciferase variant.
 5. The geneticallymodified plant cell of claim 4, wherein the luc⁺ luciferase variant isexpressed in the plant cell at a level of at least 100 pg/μg protein asdetermined by western blot analysis.
 6. The genetically modified plantcell of claim 1, wherein the bioluminescent polypeptide can be expressedin amount sufficient to produce at least one million photons of visiblelight/mm²/second.
 7. The genetically modified plant cell of claim 1,which has a reduced level of chlorophyll as compared to correspondingwild type plant cell.
 8. A transgenic plant, comprising the geneticallymodified plant cell of claim 1 or claim
 7. 9. A plant cell or tissueobtained from the transgenic plant of claim
 8. 10. A cutting of thetransgenic plant of claim
 8. 11. A seed produced by the transgenic plantof claim
 8. 12. A cDNA or genomic DNA library prepared from thetransgenic plant of claim 8, or from a plant cell or plant tissueobtained from said transgenic plant.
 13. The transgenic plant of claim8, wherein the plant is a monocot.
 14. The transgenic plant of claim 8,wherein the plant is a dicot.
 15. The transgenic plant of claim 14,wherein the plant is an angiosperm.
 16. The transgenic plant of claim15, wherein the angiosperm is a cereal plant, a leguminous plant, anoilseed plant, or a hardwood tree.
 17. The transgenic plant of claim 8,wherein the plant is an ornamental plant.
 18. The transgenic plant ofclaim 17, wherein the ornamental plant is a petunia.
 19. The transgenicplant of claim 17, wherein the ornamental plant is a carnation.
 20. Thegenetically modified plant of claim 1, wherein the heterologousnucleotide sequence encoding a bioluminescent polypeptide comprisesnucleotides 8366 to 11,113 of SEQ ID NO:2.
 21. The genetically modifiedplant of claim 1, wherein the heterologous nucleotide sequence encodinga bioluminescent polypeptide comprises nucleotides 8340 to 12,098 of SEQID NO:4 or nucleotides 6 to 3570 of SEQ ID NO:5.
 22. A recombinantnucleic acid molecule, comprising, in operative linkage, a planttranslational enhancer and a nucleotide sequence encoding abioluminescent polypeptide.
 23. The recombinant nucleic acid molecule ofclaim 22, wherein the plant translational enhancer is a plant potyvirustranslational enhancer.
 24. The recombinant nucleic acid molecule ofclaim 23, wherein the plant potyvirus is a tobacco etch virus (TEV) oran omega enhancer.
 25. The recombinant nucleic acid molecule of claim22, wherein the bioluminescent polypeptide is a luciferase polypeptideor variant thereof.
 26. The recombinant nucleic acid molecule of claim22, further comprising an operatively linked transcriptional regulatoryelement, which comprises a promoter.
 27. The recombinant nucleic acidmolecule of claim 26, wherein the transcriptional regulatory element isa cauliflower mosaic virus (CaMV) 35S regulatory element, ametallothionein regulatory element, or an actin 2 regulatory element.28. The recombinant nucleic acid molecule of claim 22, comprising, inoperative linkage, a CaMV 35S enhancer, a CaMV 35S promoter, a TEVtranslational enhancer, a nucleotide sequence encoding a luciferasepolypeptide or variant thereof, and a CaMV 35S terminator.
 29. Therecombinant nucleic acid molecule of claim 28, wherein the CaMV 35Senhancer is a dual CaMV 35S enhancer.
 30. The recombinant nucleic acidmolecule of claim 28, wherein the luciferase polypeptide or variantthereof is a luc⁺ polypeptide.
 31. The recombinant nucleic acid moleculeof claim 22, comprising, in operative linkage, a metallothionein genetranscriptional regulatory element, an omega translational enhancer, anucleotide sequence encoding a luciferase polypeptide or variantthereof, and an RbcS E9 polyA sequence.
 32. The recombinant nucleic acidmolecule of claim 22, comprising, in operative linkage, an actin 2 genetranscriptional regulatory element, an omega translational enhancer, anucleotide sequence encoding a luciferase polypeptide or variantthereof, and an RbcS E9 polyA sequence.
 33. The recombinant nucleic acidmolecule of claim 22, comprising nucleotides 8366 to 11,113 of SEQ IDNO:2.
 34. The recombinant nucleic acid molecule of claim 22, comprisingnucleotides 8340 to 12,098 of SEQ ID NO:4 or nucleotides 6 to 3570 ofSEQ ID NO:5.
 35. A vector, comprising the recombinant nucleic acidmolecule of claim
 22. 36. The vector of claim 32, wherein the vector isan expression vector.
 37. The vector of claim 35, wherein therecombinant nucleic acid molecule comprises, in operative linkage, aCaMV 35S enhancer, a CaMV 35S promoter, a TEV translational enhancer, anucleotide sequence encoding a luciferase polypeptide or variantthereof, and a CaMV 35S terminator.
 38. The vector of claim 35, whereinthe recombinant nucleic acid molecule comprises, in operative linkage, ametallothionein gene transcriptional regulatory element, an omegatranslational enhancer, a nucleotide sequence encoding a luciferasepolypeptide or variant thereof, and an RbcS E9 polyA sequence.
 39. Thevector of claim 35, wherein the recombinant nucleic acid moleculecomprises, in operative linkage, an actin 2 gene transcriptionalregulatory element, an omega translational enhancer, a nucleotidesequence encoding a luciferase polypeptide or variant thereof, and anRbcS E9 polyA sequence.
 40. The vector of claim 35, wherein therecombinant nucleic acid molecule comprises nucleotides 8366 to 11,113of SEQ ID NO:2.
 41. The vector of claim 35, wherein the recombinantnucleic acid molecule comprises nucleotides 8340 to 12, 098 of SEQ IDNO:4 or nucleotides 6 to 3570 of SEQ ID NO:5.
 42. The vector of claim35, which is selected from SEQ ID NO:1 and SEQ ID NO:2.
 43. The vectorof claim 35, which is selected from SEQ ID NO:4 and SEQ ID NO:5.
 44. Acell containing the recombinant nucleic acid molecule of claim
 22. 45. Acell containing the vector of claim
 35. 46. A method of producing agenetically modified plant cell that is visibly bioluminescent, themethod comprising introducing a transgene comprising a nucleotidesequence encoding a bioluminescent polypeptide into a plant cell,whereby the bioluminescent polypeptide is expressed at a level thatproduces at least 750,000 photons of visible light/mm²/second, therebyproducing a genetically modified plant cell that visibly bioluminesce.47. The method of claim 45, wherein the transgene further comprises aplant translational enhancer operatively linked to the nucleotidesequence encoding a bioluminescent polypeptide.
 48. The method of claim46, wherein the plant translational enhancer is a plant potyvirustranslational enhancer selected from a tobacco etch virus (TEV)translational enhancer and a tobacco mosaic virus translationalenhancer.
 49. The method of claim 46, wherein the plant translationalenhancer is an omega translational enhancer having a nucleotide sequenceset forth as nucleotides 1169 to 1235 of SEQ ID NO:5.
 50. The method ofclaim 46, wherein the bioluminescent polypeptide is a luciferasepolypeptide or variant thereof.
 51. The method of claim 47, wherein thetransgene further comprises a transcriptional regulatory elementoperatively linked to the plant translational enhancer and nucleotidesequence encoding a bioluminescent polypeptide.
 52. The method of claim51, wherein the transcriptional regulatory element is a cauliflowermosaic virus (CaMV) 35S regulatory element, a metallothionein generegulatory element, or an actin gene regulatory element.
 53. The methodof claim 46, wherein the transgene comprises, in operative linkage, adual CaMV 35S enhancer, a CaMV 35S promoter, a TEV translationalenhancer, a nucleotide sequence encoding a luciferase polypeptide orvariant thereof, and a CaMV 35S terminator.
 54. The method of claim 53,wherein the luciferase polypeptide or variant thereof is a luc⁺polypeptide.
 55. The method of claim 46, wherein the transgenecomprises, in -operative linkage, a metallothionein gene transcriptionalregulatory element, which comprises a promoter and, optionally, anenhancer; an omega translational enhancer; a nucleotide sequenceencoding a luciferase polypeptide or variant thereof; and an RbcS E9polyA sequence.
 56. The method of claim 55, wherein the luciferasepolypeptide or variant thereof is a luc⁺ polypeptide.
 57. The method ofclaim 46, wherein the transgene comprises, in operative linkage, anactin 2 gene transcriptional regulatory element, which comprises apromoter and, optionally, an enhancer; an omega translational enhancer;a nucleotide sequence encoding a luciferase polypeptide or variantthereof; and an RbcS E9 polyA sequence.
 58. The method of claim 57,wherein the luciferase polypeptide or variant thereof is a luc⁺polypeptide.
 59. The method of claim 46, wherein the transgene comprisesnucleotides 8366 to 11,113 of SEQ ID NO:2, nucleotides 8340 to 12,098 ofSEQ ID NO:4 or nucleotides 6 to 3570 of SEQ ID NO:5.
 60. The method ofclaim 46, wherein the transgene is stably maintained in the plant cellgenome.
 61. The method of claim 60, wherein the transgene is integratedinto the plant cell genome.
 62. The method of claim 46, furthercomprising contacting the plant cell, or the genetically modified plantcell derived therefrom, with an agent that inhibits carotenoidsynthesis, thereby reducing the amount of chlorophyll in the plant cell.63. The method of claim 62, wherein the agent is norfluorazon.
 64. Agenetically modified plant cell produced by the method of claim 46 orclaim
 62. 65. A transgenic plant comprising the genetically modifiedplant cell of claim
 64. 66. A genetically modified plant cell,comprising a heterologous nucleotide, which comprises, in operativelinkage, a CaMV 35S enhancer, a CaMV 35S promoter, a TEV translationalenhancer, a nucleotide sequence encoding a luciferase polypeptide orvariant thereof, and a CaMV 35S terminator wherein the luciferasepolypeptide or variant thereof can be expressed in amount sufficient toproduce at least 750,000 photons of visible light/mm²/second.
 67. Thegenetically modified plant cell of claim 66, wherein the heterologousnucleotide sequence encoding a bioluminescent polypeptide comprisesnucleotides 8366 to 11,113 of SEQ ID NO:2.
 68. A transgenic plant,comprising the genetically modified plant cell of claim 66 or claim 67.69. A plant cell or tissue obtained from the transgenic plant of claim68.
 70. A cutting of the transgenic plant of claim
 68. 71. A seedproduced by the transgenic plant of claim
 68. 72. A cDNA or genomic DNAlibrary prepared from the transgenic plant of claim 68, or from a plantcell or plant tissue obtained from said transgenic plant.
 73. Agenetically modified plant cell, comprising a heterologous nucleotidesequence, which comprises, in operative linkage, a metallothionein genetranscriptional regulatory element, an omega translational enhancer, anucleotide sequence encoding a luciferase polypeptide or variantthereof, and an RbcS E9 polyA sequence., wherein the luciferasepolypeptide or variant thereof can be expressed in amount sufficient toproduce at least 750,000 photons of visible light/mm²/second.
 74. Thegenetically modified plant cell of claim 73, wherein the heterologousnucleotide sequence comprises nucleotides 8340 to 12,098 of SEQ ID NO:475. A transgenic plant, comprising the genetically modified plant cellof claim 73 or claim
 74. 76. A plant cell or tissue obtained from thetransgenic plant of claim
 75. 77. A cutting of the transgenic plant ofclaim
 75. 78. A seed produced by the transgenic plant of claim
 75. 79. Agenetically modified plant cell, comprising a heterologous nucleotidesequence, which comprises, in operative linkage, an actin 2 genetranscriptional regulatory element, an omega translational enhancer, anucleotide sequence encoding a luciferase polypeptide or variantthereof, and an RbcS E9 polyA sequence, wherein the luciferasepolypeptide or variant thereof can be expressed in amount sufficient toproduce at least 750,000 photons of visible light/mm²/second.
 80. Thegenetically modified plant cell of claim 79, wherein the heterologousnucleotide sequence comprises nucleotides 6 to 3570 of SEQ ID NO:5. 81.A transgenic plant, comprising the genetically modified plant cell ofclaim 79 or claim
 80. 82. A plant cell or tissue obtained from thetransgenic plant of claim
 81. 83. A cutting of the transgenic plant ofclaim
 81. 84. A seed produced by the transgenic plant of claim
 81. 85. Akit, comprising the genetically modified plant cell of claim 1 or claim7, or a derivative of the genetically modified plant cell.
 86. A kitcomprising a derivative of the genetically modified plant cell, whereinthe derivative of the genetically modified plant cell is a transgenicplant comprising the genetically modified plant cell of claim 1 or claim7, or a cell, tissue or organ of said transgenic plant.
 87. The kit ofclaim 86, wherein the organ of said transgenic plant is a flower or abract.
 88. The kit of claim 85, further comprising an amount ofluciferin sufficient for generating visible bioluminescence of thegenetically modified plant cell or the derivative of the geneticallymodified plant cell.
 89. The kit of claim 85, further comprising anamount of a carotenoid inhibitor sufficient for reducing or inhibitingchlorophyll production in the genetically modified plant cell or thederivative of the genetically modified plant cell.
 90. A kit, comprisinga cutting or a seed of the transgenic plant of claim
 8. 91. The kit ofclaim 90, further comprising reagents for growing a transgenic plantfrom the cutting or seed.
 92. The kit of claim 90, further comprising anamount of a carotenoid inhibitor sufficient for reducing or inhibitingchlorophyll production in a transgenic plant grown from the cutting orseed.
 93. The kit of claim 90, further comprising an amount of luciferinsufficient for generating visible bioluminescence of a transgenic plantgrown from the cutting or seed.
 94. A kit, comprising the geneticallymodified plant cell of claim 66, 67, 73, 74, 79, or 80, or a derivativeof the genetically modified plant cell.
 95. A kit comprising aderivative of a genetically modified plant cell, wherein the derivativeof the genetically modified plant cell is a transgenic plant comprisingthe genetically modified plant cell of claim 66, 67, 73, 74, 79 or 80,or a cell, tissue or organ of said transgenic plant.
 96. The kit ofclaim 95, wherein the organ of said transgenic plant is a flower or abract.
 97. The kit of claim 94, further comprising an amount ofluciferin sufficient for generating visible bioluminescence of thegenetically modified plant cell or the derivative of the geneticallymodified plant cell.
 98. The kit of claim 94, further comprising anamount of a carotenoid inhibitor sufficient for reducing or inhibitingchlorophyll production in the genetically modified plant cell or thederivative of the genetically modified plant cell.
 99. A kit, comprisinga cutting of the transgenic plant of claim 68, or a seed produced bysaid transgenic plant.
 100. The kit of claim 99, further comprisingreagents for growing a transgenic plant from the cutting or seed. 101.The kit of claim 99, further comprising an amount of a carotenoidinhibitor sufficient for reducing or inhibiting chlorophyll productionin a transgenic plant grown from the cutting or seed.
 102. The kit ofclaim 99, further comprising an amount of luciferin sufficient forgenerating visible bioluminescence of a transgenic plant grown from thecutting or seed.
 103. The kit of claim 95, further comprising an amountof luciferin sufficient for generating visible bioluminescence of thegenetically modified plant cell or the derivative of the geneticallymodified plant cell.
 104. The kit of claim 95, further comprising anamount of a carotenoid inhibitor sufficient for reducing or inhibitingchlorophyll production in the genetically modified plant cell or thederivative of the genetically modified plant cell.
 105. A kit comprisinga cutting of the transgenic plant of claim 75, or a seed produced bysaid transgenic plant.
 106. The kit of claim 105, further comprisingreagents for growing a transgenic plant from the cutting or seed. 107.The kit of claim 105, further comprising an amount of a carotenoidinhibitor sufficient for reducing or inhibiting chlorophyll productionin a transgenic plant grown from the cutting or seed.
 108. The kit ofclaim 105, further comprising an amount of luciferin sufficient forgenerating visible bioluminescence of a transgenic plant grown from thecutting or seed.
 109. A kit comprising a cutting of the transgenic plantof claim 81, or a seed produced by said transgenic plant.
 110. The kitof claim 109, further comprising reagents for growing a transgenic plantfrom the cutting or seed.
 111. The kit of claim 110 further comprisingan amount of a carotenoid inhibitor sufficient for reducing orinhibiting chlorophyll production in a transgenic plant grown from thecutting or seed.
 112. The kit of claim 109, further comprising an amountof luciferin sufficient for generating visible bioluminescence of atransgenic plant grown from the cutting or seed.